Rubyrin macrocycles

ABSTRACT

The present invention is directed to the synthesis and use of novel macrocyclic compounds, based upon a new class of expanded porphyrins, termed rubyrins. Disclosed herein is the structure and synthesis of a prototypical rubyrin and various substituted rubyrin analogues, conjugates and compositions. Rubyrin itself is characterized by the presence of six pyrrolic subunits contained within a fully aromatic 26 π-electron macrocyclic framework and by UV/VIS absorption bands that are very red-shifted as compared to those of other porphyrins or pentapyrrolic expanded porphyrins. The rubyrin-type class of compounds is further characterized by an ability to undergo facile protonation at the pyrrolic nitrogens and, once protonated, by an ability to form complexes with anions such as nucleotide-containing compounds. Rubyrin-based compounds are useful as, for example, anion chelators and receptors and as transporters for various anionic compounds including antiviral agents. In addition to the 26 π-electron target system, the present invention concerns other oxidation states bearing the same connectivity and the same total number of non-hydrogen atoms, and various analogues in which different substituents are present at the various meso and/or β-pyrrolic positions or in which furan and/or thiophene moieties replace one or more of the six pyrrolic subunits.

This application is a divisional of U.S. Ser. No. 08/015,208, filed Feb.9, 1993, now U.S. Pat. No. 5,410,045 which is a continuation-in-part ofU.S. Ser. No. 07/926,357, filed Aug. 4, 1992, now abandoned. Thegovernment owns rights in the present invention pursuant to NIH grant AI28845.

BACKGROUND OF THE INVENTION

1. Field of the. Invention

The present invention relates generally to macrocyclic expandedporphyrin compounds, and particularly, to the novel expanded porphyrintermed rubyrin and to a class of rubyrin analogues. Disclosed arerubyrin and rubyrin analogue compounds compositions, and methods ofusing protonated rubyrin compounds as anion chelators, receptors andtransporters.

2. Description of the Related Art

In recent years increasing effort has been devoted to the preparation ofnovel "expanded porphyrins"¹, large pyrrole-containing macrocyclicanalogues of the porphyrins (e.g. porphine, structure 1, FIG. 1) and anumber of such systems are now known¹⁻¹⁷. However, only a few fullyconjugated examples have been reported that contain more than fourpyrrolic subunits, namely the smaragdyrins²,3, sapphyrins²⁻⁶,pentaphyrins⁷⁻⁸, hexaphyrins⁹, and superphthalocyanines¹⁰. Thesecompounds are represented in their generalized substituent-free forms asstructures 2-6 (FIG. 1).

To date, there remains relatively little documented informationconcerning the chemistry of the above-mentioned expanded porphyrinsystems. Indeed, at present, structural information is available onlyfor derivatives of sapphyrin (e.g. structure 2)⁴,5 and pentaphyrin (e.g.structure 3)⁸ in the all-pyrrole series. Therefore, numerous fundamentalquestions concerning these molecules still remain to be answered, suchas those pertaining to ring size, aromaticity, and effective macrocyclestability. The synthesis and structural characterization of hexapyrrolicmacrocycles would be a particular advance in this area, allowing theanswers to such inter-related questions to be elucidated.

It has long been appreciated that a considerable number of ionic (e.g.phosphorylated) nucleotide analogues exhibit antiviral activity incell-free extracts, yet are inactive in vivo due to their inability tocross lipophilic cell membranes²⁵,26. For example, the anti-herpeticagent, acyclovir (FIG. 9a; 9-[(2-hydroxyethoxy)methyl]-9H-guanine), istypical in that it is able to enter the cell only in its unchargednucleoside-like form. Upon gaining entry to the cytoplasm it isphosphorylated, first by a viral-encoded enzyme, thymidine kinase (FIG.9b), and then by relatively nonspecific cellular enzymes to produce theactive, ionic triphosphate nucleotide-like species (FIG. 9c). There itfunctions both as an inhibitor of the viral DNA polymerase and as achain terminator for newly synthesized herpes simplex DNA.

Many other potential antiviral agents, including, for instance, theanti-HIV agent, Xylo-G (FIG. 9d; 9-(β-D-xylofuranosyl)quanine), on theother hand, are not phosphorylated by a viral enzyme and are, therefore,largely or completely inactive²⁷. If, however, the activemonophosphorylated forms of these putative drugs (such as in FIG. 9e)could be transported into cells, it would be possible to fight viralinfections with a large battery of otherwise inactive materials. If suchspecific into-cell transport were to be achieved, it would thereforegreatly augment the treatment of such debilitating diseases as, forexample, AIDS, herpes, hepatitis and measles. Given the fact that AIDSis currently a major national health problem of frightening proportions,and that something so nominally benign as measles still claims over100,000 lives per year world-wide²⁶, treatment of these diseases wouldbe particularly timely and worthwhile.

At present, no general set of nucleotide transport agents exists. Inearly work, Tabushi was able to effect adenosine nucleotide transportusing a lipophilic, diazabicyclooctane-derived, quaternary aminesystem²⁸. However, this same system failed to mediate the transport ofguanosine 5'-monophosphate (GMP) or other guanosine-derived nucleotides.Since then, considerable effort has been devoted to the generalizedproblem of nucleic acid base ("nucleobase") recognition, and variousbinding systems have been reported.

Currently known nucleotide binding systems include acyclic, macrocyclic,and macrobicyclic polyaza systems²⁹⁻³⁷, nucleotide-bindingbis-intercalands³⁸ ; guanidinium-based receptors³⁹⁻⁴⁶ ; and variousrationally designed H-bonding receptors⁴⁷⁻⁵³. These latter H-bondingreceptors have been shown to be effective for the chelation of neutralnucleobase and/or nucleoside derived substrates but, without exception,have also all proved unsatisfactory for the important task of chargednucleotide recognition. Large macrocyclic compounds, particularlymacrocyclic compounds larger than sapphyrins, which could be relativelyeasily protonated could prove to be useful in anion binding andtransport.

Despite intensive efforts in this field, there is currently no syntheticsystem capable of effecting the recognition and through-membranetransport of phosphate-bearing species such as anti-viral compounds.Furthermore, there are presently no rationally designed receptors whichare "tunable" for the selective complexation of a givennucleobase-derived system.

There is clearly, therefore, a major need for novel drug deliverysystems to be developed. Compounds which would allow negatively-charged(anionic) structures, particularly specifically-recognized nucleotides,to be transported across naturally lipophilic cellular membranes wouldrepresent an important scientific and medical advance. The developmentof such anion carriers may also prove to be important in the clinicaltreatment of cystic fibrosis, in that such compounds would likelyfacilitate the out-of-cell diffusion of chloride anions.

SUMMARY OF THE INVENTION

The present invention addresses these and other shortcomings in theprior art by providing compositions for use in specific anion bindingand transport. The invention concerns a class of novel expandedporphyrins, or macrocycles, termed rubyrin and analogues thereof.Rubyrin and rubyrin analogues are able to bind negatively chargedsubstances, anions, at near-neutral pH, and have the ability totransport anionic compounds across cell membranes. The rubyrinmacrocyclic compounds of this invention are particularly contemplatedfor use transporting antiviral nucleotide analogues into cells, and infacilitating chloride anion exit from cells. They will thus find use inthe treatment of a variety of viral diseases and also cystic fibrosis.

Rubyrin and substituted derivatives thereof are red in dilute organicsolution, hence the use of the trivial name "rubyrin" from the Latinrubeus, for this new class of expanded porphyrins. Rubyrin itself is amacrocycle which can be generally characterized by the presence of sixpyrrolic subunits contained within a fully aromatic 26 π-electronmacrocyclic framework and by UV/VIS absorption bands that are veryred-shifted as compared to those of either porphyrins or sapphyrins(pentapyrrolic expanded porphyrins).

In a general and overall sense, the novel rubyrin compounds of thepresent invention include those with structures in accordance with thegeneral structures I, II and III, as set forth in FIG. 2. In thesestructures, the substituents A₁ and A₂ may be nitrogen, oxygen orsulphur. In general, the substituents R₁, R₂, R₃, R₄, R₅ and R₆ and X₁,X₂, X₃, and X₄ may separately and independently be H, alkyl, aryl,amino, hydroxyl, alkoxy, carboxy, carboxamide, ester, amide, sulfonato,hydroxy substituted alkyl, alkoxyl substituted alkyl, carboxysubstituted alkyl, amino substituted alkyl, sulfonato substituted alkyl,ester substituted alkyl, amide substituted alkyl, substituted aryl,substituted alkyl, substituted ester, substituted ether or substitutedamide.

In certain embodiments, any one of the substituents R₁ -R₆ or X₁ -X₄ maybe of the formula (CH₂)_(n) --A--(CH₂)_(m) --B. In this formula, n and mare integers<10 or zero; A may be CH₂, O, S, NH or NR₇, wherein R₇ mayagain be any of the groups listed above for R₁ -R₆ ; and B will be achosen, or desirable, functional unit. A "chosen functional unit" isused herein to refer to any compound or substance which one may desireto conjugate to a rubyrin molecule. Compounds based upon nucleobases,such as a single nucleobase, modified nucleobase, a nucleobase-typeanti-viral compound, or an oligo- or polynucleotide; or compounds basedupon saccharides, such as a sugar, sugar derivative or polysaccharide,are particularly contemplated for use in conjugating to rubyrin.However, the use of other compounds in rubyrin conjugates is alsoenvisioned. Further suitable compounds include, for example, metalchelating groups; alkylating agents; steroids and steroid derivatives;amino acids, peptides and polypeptides; further rubyrin molecules,rubyrin derivatives or polymeric rubyrins; other macrocyclic compoundsuch as sapphyrin or texaphyrin, or polymers or derivatives thereof; oreven a polymeric matrix or solid support.

Rubyrin and analogues thereof may be further characterized by theability to undergo facile protonation at the pyrrolic nitrogens and,once protonated, by an ability to form complexes with anions. Anadvantageous functional characteristic of rubyrin and analogues thereofis the ability to bind anions at near-neutral pH and to transport anionsacross lipophilic structures such as biological membranes. Preferredcompounds of the present invention therefore have the capacity to bindanions and yet the ability to retain overall supramolecular chargeneutrality. In particular, the fact that rubyrins are larger thansapphyrins, the only other class of compounds known to display suchbehavior, makes them considerably easier to protonate and thus much moreeffective in the recognition and transport of anions than this otherclass of molecules. This then represents a significant advance embodiedin the present invention.

In certain embodiments, the invention concerns a macrocyclic rubyrincompound, represented in its generalized substituent free form bystructure 7 (FIG. 2), a structure in turn that corresponds to themacrocyclic system29,30,31,32,33,34-hexaazaheptacyclo[24.2.1.1²,5.1⁷,10.1¹²,15.1¹⁶,19.1²¹,24]tetratriaconta-1,3,5,7(31),8,10,12,14,16,18,20,22,24(34),25,27-pentadecaene.

It will be understood by those of skill in the art of organic chemistry,however, that the present invention is not limited to compounds inaccordance with structure 7. Indeed, also included within the context ofthis disclosure are analogues of 7, namely macrocycles based uponstructures 8 and 9, which have the same overall connectivity and samenumber of non-hydrogen atoms but which differ from the structurallycharacterized 26 π-electron prototypes by the number of total electronscontained within the π-electron periphery. For example, 28π-electronrubyrin analogues, as described in Examples V and VI, fall within thescope of the present invention. A particular example of a compound inaccordance with structure 8 is structure 19 of FIG. 5, namely4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethyl-29,30,31,32,33,34-hexaazaheptacyclo[24.2.1.1²,5.1⁷,10.1¹²,15.1¹⁶,19.1²¹,24]tetratriaco nta-1,3,5,7,9,11,13,15,17,19,21,23,25,27-tetradecaene.

Rubyrin compounds in which two or more of the pyrrolic nitrogen atomshave been replaced by oxygen or sulfur are also encompassed by thepresent invention. In this context, the oxygen or sulfur atoms aretermed "heteroatoms", and the resultant rubyrin analogues may bereferred to as heteroatomic compounds. Rubyrin analogues comprising fourheteroatoms are exemplified in general by the macrocycle structures20-24 (FIG. 6), which may variously contain two or four nitrogen, oxygenor sulphur atoms. The synthesis of rubyrin analogues in which two, four,or even six, of the nitrogen atoms have been replaced by oxygen orsulfur is described in Example X. The condensation of differing oxygen-or sulphur-containing units, from any one of a variety of readilyavailable starting materials, under the rubyrin forming conditionsdisclosed herein, will result in the generation of macrocycles with anydesired combination of nitrogen, oxygen or sulfur, based on startingmaterials chosen. Macrocycles containing combinations of nitrogen witheither oxygen or sulphur, or alternatively, oxygen in combination withsulphur, such as those represented by structures 87, 92, 99 and 104 inreaction schemes P through S, are therefore encompassed by the presentinvention.

It will also be understood that any of the above rubyrin compounds maybe either singly or doubly protonated, and in certain embodiments,triply or four-fold protonated.

The rubyrin compounds of the present invention are specificallyexemplified by those compounds having structures in accordance withstructures 10a, 10b, 17a, 17b, 18a, 18b and 19, as set forth in FIGS. 3,4 and 5, respectively, as well as by other structures discussedhereinbelow and/or set forth in the figures and various reaction schemespresented herein. As will be understood by those of skill in the art,the present invention also encompasses several other rubyrin analogues.For example, different combinations of bipyrroles and pyrroles may beemployed, such as those set forth in FIGS. 11 and 12, both in the firstand second condensation steps, to yield a variety of different products.In one instance, this is exemplified herein by the generation of arubyrin analogue in which four of the 12 possible β-pyrrolic positionsare either unsubstituted or replaced by a range of functionalized alkylgroups. The generation of various other rubyrin analogues is describedthroughout the specification, such as in Example VII, and represented invarious reaction schemes, for example, Schemes A through E. A range ofcompounds with a wide variety of alkyl and/or aryl substituents in themeso and/or β-pyrrolic positions thus fall within the scope of thepresent invention, as represented by exemplary structures 27, 35, 40 and47.

Any macrocyclic compound with a structure in accordance with any ofstructures 7, 8, 9, or 20-24 may be prepared in which differentsubstituents are present at the various meso and/or β-pyrrolic positionsor in which furan and/or thiophene moieties replace one or more of thesix pyrrolic subunits. Such substituents may separately andindependently include, H, alkyl, aryl, amino, hydroxyl, alkoxy, carboxy,carboxamide, ester, amide, sulfonato, hydroxy substituted alkyl, alkoxylsubstituted alkyl, carboxy substituted alkyl, amino substituted alkyl,sulfonato substituted alkyl, ester substituted alkyl, amide substitutedalkyl, substituted aryl, substituted alkyl, substituted ester,substituted ether, or substituted amide groups.

Examples of functional group manipulation in β-substituted pyrroles areshown in FIG. 11, and starting pyrroles in FIG. 12. Methods for thepreparation of such pyrroles will be apparent to those of skill in theart and are described in various references, for example, Paine(1978)^(81a) ; Sessler et al. (1991)^(81b) ; and Zard & Barton,(1985)^(81c). Functional group manipulation of the B-positions ispossible after macrocycle formation, or the appropriately substitutedpyrroles may be used directly, depending upon the particular pyrrole tobe used. The α-positions may be carboxyl-, ester-, formyl-, alkyl-,and/or unsubstituted and may also be directly linked to other pyrrolesor linked to other pyrroles via a methylene bridge.

The synthetic methodology of the present invention also provides for thesynthesis of a wide variety of other rubyrin derivatives or conjugates,wherein a functionalized rubyrin macrocycle is appended to a moiety ofdesirable chemical function. Rubyrin may thus also be conjugated to, forexample, nucleobases, nucleobase derivatives and oligo- orpolynucleotides; sugars, sugar derivatives and polysaccharides; metalchelating agents; other rubyrin molecules, polyrubyrins, sapphyrin ortexaphyrin, polymers and solid support matrices; amino acids, peptidesand polypeptides; steroids and steroid derivatives; and alkylatingagents. It is contemplated that one of skill in the art will be able toprepare various rubyrin conjugates, including those listed above,without undue experimentation given the readily-available startingmaterials and in light of the synthetic methodology disclosed in thepresent application.

In certain embodiments, rubyrin compounds are contemplated which containthe rubyrin macrocycle core for phosphate binding and also nucleobase"appendages" for specific nucleic acid recognition (FIG. 10). These arereferred to as rubyrin-nucleobase conjugates, which term is intended toinclude any conjugate formed by the covalent conjugation of any rubyrinmacrocycle to any nucleobase. The rubyrin-nucleobase conjugates of thepresent invention may be of either the mono- or di-substituted forms, asrepresented by the general structures IV and V (FIG. 10). Amono-substituted rubyrin-nucleobase conjugate is a compound in whichonly one position, i.e., only one of the groups R₁ -R₆, or even X₁ -X₄,is covalently attached to a nucleobase-containing compound, whereasdi-substituted conjugates have nucleobase substitutions at twoindependent positions. Both mono- and di-substituted conjugates maycomprise one, two, or a plurality of nucleobase units, the differencebeing in the point of attachment and not in the number of individualunits attached to the macrocycle. The synthesis of such conjugates isdescribed in Example VIII and specific rubyrin-nucleobase conjugates areexemplified in structures 51, 55, 59 and 63 of reaction schemes Fthrough I.

The term "nucleobase" as used herein, refers generally to any moietythat includes within its structure a purine or pyrimidine, a nucleicacid, nucleoside, nucleotide, or any derivative of any of these, such asa "protected" nucleobase. Thus, the term nucleobase includes adenine,cytosine, guanine, thymidine, uridine, inosine, and the like, bases,nucleotides or nucleosides, as well as any base, nucleotide ornucleoside derivative based upon these or related structures.

A particular example of a useful nucleobase are the so-calledantimetabolites that are based upon the purine or pyrimidine structure.These structures typically exert their biological activity asantimetabolites through competing for enzyme sites and therebyinhibiting vital metabolic pathways. However, in the context of thepresent invention, the inventors are employing the term "antimetabolitenucleobase" quite broadly to refer to any purine or pyrimidine-basedmolecule that will effect an anticellular, antiviral, antitumor,antiproliferative or antienzymatic effect, regardless of the underlyingmechanism. Exemplary structures are shown in Table 1, and include theantimetabolites FU, AraC, AZT, ddI, xylo-GMP, Ara-AMP, PFA and COMDP.

It is contemplated that rubyrin-nucleobase conjugates will have a widevariety of applications, including their use as carriers for thedelivery of antiviral drugs to a particular body or even subcellularlocale. In the case of antimetabolite nucleobases, it is known that manynucleobase antimetabolites can not be readily employed in therapy due tothe fact that their charged nature inhibits their uptake by targetcells, or otherwise inhibits or suppresses their unencumbered movementacross biological membranes. Typically, this shortcoming is due to thepresence of charged structures such as phosphates, phosphonates,sulfates or sulfonates on the nucleobase, which due to their chargednature prevents or inhibits their crossing of a biological membrane. Itis proposed that the rubyrins of the present invention can be employedas transport agents for carrying such nucleobases across membranes,(whether the nucleobase is directly conjugated to the macrocycle orsimply complexed with it). This point is elaborated in further detail inSessler et al (1992)²⁴ incorporated herein by reference.

Generally speaking, in the context of rubyrin-nucleobase constructsdesigned for drug delivery it will usually be the case that one willemploy only one or two nucleobase-containing substituents for eachrubyrin macrocycle. Rubyrin derivatives with a single nucleobase aretermed "ditopic receptors", whereas those with two nucleobases aretermed "tritopic receptors". However, the invention is not limited tocompounds containing one or two nucleobase units, indeed, mono- ordi-substituted rubyrin-nucleobase conjugates may have any number ofnucleobases or nucleobase oligomers or polymers attached. The ultimatenumber of such residues that are attached will, of course, depend uponthe application. One may employ a rubyrin derivative with 10 or so basesattached to bind and transport complementary oligo- or poly-nucleotides.Of course, there is no limitation to the particular position(s) withinthe rubyrin macrocycle to which the nucleobase(s) may be attached tocreate a conjugate.

Further examples of rubyrin derivatives or conjugates encompassed by thepresent invention are rubyrin saccharide derivatives, wherein themacrocycle is appended to a saccharide-based unit, such as a sugar,sugar derivative or polysaccharide. The synthesis of rubyrin-saccharidecompounds is described in Example IX and specific rubyrin saccharideconjugates are represented by structures 66, 68, 76 and 84 in reactionschemes J, K, M and O, respectively. A non-exhaustive, exemplary list ofsugars which may be conjugated to rubyrin in this manner is set forth inTable 2. Of course, any sugar or modified sugar may be employedincluding sugars having additional phosphate, methyl or amino groups andthe like. Moreover, the use of both D- and L-forms, as well as the α andβ forms are also contemplated. The use of sugars such as glucose,galactose, galactosamine, glucosamine and mannose is particularlycontemplated. Rubyrin-saccharide conjugates are envisioned to be of usewhere one would like to control, or otherwise modify, the watersolubility of the resultant rubyrin-based compound, for example, inexploiting its novel properties in connection with human or animalapplications.

In still further embodiments, the invention concerns compositions whichare composed of a rubyrin derivative in accordance with any one of theembodiments discussed above complexed to a second substance, wherein thesecond substance includes within its structure a negatively chargedmoiety. More particularly, the second substance will include anegatively charged component such as a chloride, phosphate, phosphonate,sulfate, or sulfonate moiety, of which, rubyrin-chloride ion complexesare a particular example.

In other preferred embodiments, the second substance will include apurine or pyrimidine, or an analog of either, within its structure. Asmentioned above, these nucleobase structures include, for example,adenine, cytosine, guanine, thymidine, uridine and inosine; variousantimetabolic or antienzymatic nucleobase compounds; and also oligo orpolynucleotides such as DNA or RNA. Antimetabolic and antienzymaticcompounds include those with antitumor, anticellular, antiproliferativeand antiviral activity, examples of which are represented in Table 1.

In further related embodiments, the invention concerns a method forforming a complex between a rubyrin macrocycle and a negatively chargedsecond substance, or selected agent. The method involves preparing arubyrin or rubyrin analogue or conjugate, such as any one of the rubyrinderivatives as described above, and contacting this rubyrin or rubyrinderivative with a negatively charged substance or selected agent underconditions effective to allow the formation of a complex between therubyrin macrocycle and the negatively charged substance.

This method is appropriate for binding, or complexing, a range ofnegatively charged substances or selected agents, such as for example,chloride ions and other halides, pseudohalides such as azide or cyanideanions, and anionic clusters such as ferricyanide. The complexing ofphosphate-containing compounds, including, simple alkyl or arylphosphates, nucleotides, oligo- and polynucleotides, such as DNA, RNAand anti-sense constructs, and nucleotide analogues is particularlycontemplated. Even more preferable, is the complexing of antiviralcompounds such as phosphonate derivatives and simple species such as thepyrophosphate derivatives PFA and COMDP; the antiviral agents of FIG.9b,e,f,g,h,i, and Table 1, and particularly, acyclovir monophosphate,Xylo-GMP, Ara-AMP. Here, as throughout this invention, a key embodimentis the high basicity of the rubyrin or pseudo rubyrin core, which allowsthis class of compounds and its derivatives to act as vastly improvedanion chelators or carriers in comparison to expanded porphyrins such assapphyrins or any other extant system.

Still further methodological embodiments concern methods for thecellular transport of a given substance, generally a negatively chargedsubstance. This may be employed as a means of, for example, successfullyintroducing a negatively charged substance into a cell, oralternatively, as a means of facilitating the removal of a negativelycharged substance from a cell. To achieve this, one would prepare arubyrin macrocycle in accordance with the present invention, contact themacrocycle with the negatively charged substance under conditionseffective to allow complex formation, and then simply contact the cell,either in vitro or in vivo, with the macrocycle-bound substance.

Any one of a variety of negatively charged substances may be deliveredto a cell, including a cell within an organism, in this manner. Thedelivery of polynucleotides, including anti-sense constructs, andnucleotide analogues, such as antiviral compounds, is particularlyenvisioned. One example concerns the introduction of a rubyrin-complexcomposition which includes an antiviral antimetabolic or antienzymaticcompound into a cell suspected of being a virally infected target cell.Another example is the introduction of an antitumor antimetabolic orantienzymatic compound into a cell suspected of being a tumor orproliferating cell.

Of course, it is contemplated that such target cells may be locatedwithin an animal or human patient, in which case an effective amount ofthe complex, in pharmacologically acceptable form, would be administeredto the patient. Generally speaking, it is contemplated by the inventorsthat useful pharmaceutical compositions of the present invention willinclude the selected rubyrin derivative in a convenient amount that isdiluted in a pharmacologically or physiologically acceptable buffer,such as, for example, phosphate buffered saline. The route ofadministration and ultimate amount of material that is administered tothe patient or animal under such circumstances will depend upon theintended application and will be apparent to those of skill in the artin light of the examples which follow. Preferred routes ofadministration will typically include parenteral or topical routes.

The capacity of rubyrin and analogues thereof to effect specificinto-cell transport of anti-viral compounds is contemplated to be of useagainst a wide variety of debilitating diseases such as AIDS, herpes,hepatitis and measles. As a mediator of DNA import, rubyrins mayconceivably be employed in the treatment of any disease in which thedelivery of an oligonucleotide or DNA fragment would be advantageous,such as in supplying a functioning gene, or in inhibiting an aberrantgene, for example, by employing an antisense DNA construct. As discussedabove, the larger size, high basicity, and relative ease with whichrubyrins may be protonated, renders them particularly effectivemolecules for use in anion transport.

Additionally, certain rubyrins with advantageous chloride iontransporting properties may be employed as synthetic carriers capable offacilitating out-of-cell diffusion of chloride anions, and are thereforecontemplated for use as therapeutic agents for the treatment of cysticfibrosis.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Porphyrins and expanded porphyrins (large pyrrole-containingmacrocyclic porphyrin analoguss). Structure 1, porphine; structure 2,smaragdyrin; structure 3, sapphyrin; structure 4, pentaphyrin; structure5, hexaphyrin; structure 6, superphthalocyanine. Compounds in structures2-6 are all represented in their generalized substituent-free forms.

FIG. 2. Rubyrin and rubyrin analogues. Structure 7, rubyrin(29,30,31,32,33,34-hexaazaheptacyclo[24.2.1.1²,5.1⁷,10.1¹²,15.1¹⁶,19.1²¹,24]tetratriaconta-1,3,5,7(31),8,10,12,14,16,18,20,22,24(34),25,27-pentadecaene);structures 8 and 9, the same overall connectivity and same number ofnon-hydrogen atoms as rubyrin, but different number of total electronscontained within the y-electron periphery. Structures I, II and IIIrepresent more general versions of the rubyrins of the presentinvention. In these structures, A₁ and A₂ may be nitrogen, oxygen orsulphur. The substituents R₁ -R₆ and X₁ -X₄ may be separately andindependently H, alkyl, aryl, amino, hydroxyl, alkoxy, carboxy,carboxamide, ester, amide, sulfonato, hydroxy substituted alkyl, alkoxylsubstituted alkyl, carboxy substituted alkyl, amino substituted alkyl,sulfonato substituted alkyl, ester substituted alkyl, amide substitutedalkyl, substituted aryl, substituted alkyl, substituted ester,substituted ether, substituted amide; or may be of the formula (CH₂)_(n)--A--(CH₂)_(m) --B. In this formula, n and m are integers<10 or zero; Amay be CH₂, O, S, NH or NR₇, wherein R₇ may again be any of the abovegroups; and B will be a chosen or selected functional group. Preferredgroups for B are contemplated to be nucleobases, modified nucleobases,oligonucleotides, sugars, sugar derivatives or polysaccharides; however,other suitable groups include, for example, metal chelating groups,alkylating agents, steroids, steroid derivatives, amino acids, peptides,polypeptide, rubyrin, rubyrin derivative, polymeric rubyrin, othermacrocyclic compounds such as sapphyrins or texaphyrins, polymericmatrices or solid supports.

FIG. 3. Structure 10a,4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethyl-29,30,31,32,33,34-hexaazaheptacyclo[24.2.1.1²,5.1⁷,10.1¹²,15.1¹⁶,19.1²¹,24]tetratriaconta-1,3,5,7(31),8,10,12,14,16,18,20,22,24(34),25,27-pentadecaene(4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethylrubyrin); structure10b, the counterpart wherein R₁ =CH₃ and R₂ =H.

FIG. 4. Reaction scheme for the synthesis of structure 17, thediprotonated form of4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethylrubyrin, alsodemonstrating structures 11-16; 17a, 17b, 18a and 18b.

FIG. 5. Structure 19, the rubyrin-like system,4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethyl-29,30,31,32,33,34-hexaazaheptacyclo[24.2.1.1²,5.1⁷,10.1¹²,15.1¹⁶,19.1²¹,24 ]tetratriaconta-1,3,5,7,9,11,13,15,17,19,21,23,25,27-tetradecaene.

FIG. 6. Structures 20-24, rubyrin analogues employing heteroatoms, i.e.,variously including nitrogen, oxygen and sulphur atoms. In that thesestructures are generally based upon structures I, II and III, it will beunderstood that they may also include any of the substituents listedabove for R₁ -R₆ and X₁ -X₄, namely, H, alkyl, aryl, amino, hydroxyl,alkoxy, carboxy, carboxamide, ester, amide, sulfonato, hydroxysubstituted alkyl, alkoxyl substituted alkyl, carboxy substituted alkyl,amino substituted alkyl, sulfonato substituted alkyl, ester substitutedalkyl, amide substituted alkyl, substituted aryl, substituted alkyl,substituted ester, substituted ether and substituted amide.

FIG. 7. The UV/VIS absorption spectra of 17b (--) and the diprotonatedderivatives of 2,3,7,8,12,13,17,18-octaethylporphyrin (recorded in thepresence of excess TFA) (.sup.. . . ) and3,8,12,13,17,22-hexaethyl-2,7,18,23-tetramethylsapphyrin (recorded asthe dihydrochloride salt) (--) in dilute CH₂ Cl₂.

FIG. 8. Molecular structure of 17b showing partial atom labeling schemeand the H-bonding interactions of the Cl⁻ counterions with themacrocycle (dashed lines). Top: View perpendicular to the plane throughthe nitrogen atoms. Below: Side view showing the nearly planarconformation of the core macrocycle. The macrocycle lies around aninversion center. Atoms labeled with a' are related by -x,1-y, 1-z.Thermal ellipsoids are scaled to the 30% probability level. H atomsrepresented as spheres of arbitrary size. The disordered atoms and thechloroform molecules are not shown. Selected bond distances (Å) andangles (°): N1 .sup.. . . C1, 1.373(5); N1 .sup.. . . C4, 1.394(6); C1.sup.. . . C2, 1.437(7); C2 .sup.. . . C3, 1.362(5); C3 .sup.. . . C4,1.429(6); C4 .sup.. . . C5, 1.381(5); C5 .sup.. . . C6, 1.379(6); C6.sup.. . . C7, 1.421(5); C7 .sup.. . . C8, 1.377(9); C8 .sup.. . . C9,1.449(8); N2 .sup.. . . C6, 1.383(7); N2 .sup.. . . C9, 1.367(5); N1.sup.. . . N2, 3.483(5); N1 .sup.. . . N3, 5.559(5); N1 .sup.. . . N1',6.352(7); N1 .sup.. . . N2', 5.836(5); N1.sup.. . . N3', 3.078(5);N2.sup.. . . N2', 7.213(7); N3 .sup.. . . N3'6.354(7); N1 .sup.. . .C11, 3.265(4); N2 .sup.. . . C11, 3.158(4); N1 .sup.. . . C4 .sup.. . .C5, 130.1(4); C3 .sup.. . . C4 .sup.. . . C5, 123.4(4); C4 .sup.. . . C5.sup.. . . C6, 137.6(5); C5 .sup.. . . C6 .sup.. . . C7, 122.9(5); C5.sup.. . . C6 .sup.. . . N2, 129.5(4).

FIG. 9. Structures of anti-viral and potentially anti-viral compounds a,acyclovir (9-[(2-hydroxyethoxy)methyl]-9H-guanine); b, phosphorylatedform after viral thymidine kinase action; c, active, ionic triphosphatenucleotide-like species; d, Xylo-G (9-(β-D-xylofuranosyl)guanine); e,phosphorylated form; f & g, anti-HSV and anti-HIV phosphonatederivatives; h, the pyrophosphate derivative PFA; and i, thepyrophosphate derivative COMDP.

FIG. 10. Rubyrin nucleobase derivatives. Rubyrin nucleobase conjugatesmay be of the mono- or di-substituted forms, as represented by thegeneral structures IV and V. Mono-substituted rubyrin-nucleobaseconjugates may contain, but are not limited to, a single nucleobaseunit, when they are termed ditopic rubyrin receptors. Di-substitutedrubyrin-nucleobase conjugates containing two nucleobase units are termedtritopic rubyrin receptors. In structures IV and V, the groups R₁ -R₆which do not contain a nucleobase functional unit, and groups X₁ -X₄,may be H, alkyl, aryl, amino, hydroxyl, alkoxy, carboxy, carboxamide,ester, amide, sulfonato, hydroxy substituted alkyl, alkoxyl substitutedalkyl, carboxy substituted alkyl, amino substituted alkyl, sulfonatosubstituted alkyl, ester substituted alkyl, amide substituted alkyl,substituted aryl, substituted alkyl, substituted ester, substitutedether and substituted amide. At least one of the R groups will be of theformula (CH₂)_(n) --A--(CH₂)_(m) --B, wherein A may be CH₂, O, S, NH orNR₇, and R₇ may be any of the groups listed above and B may be one ormore nucleobases, nucleobase derivatives or protected nucleobases.Conjugation of a nucleobase to a rubyrin derivative to form amononucleobase rubyrin conjugate may be via any of the R or X groups.Conjugation of the two separate nucleobases to a rubyrin derivative toform a dinucleobase rubyrin conjugate may also be via any two of thesegroups, however, it is contemplated that the creation of a symmetricalmolecule will generally be preferred. The rubyrin nucleobase derivativesmay include any purine or pyrimidine nucleobase, such as cytosine,guanine, thymidine, adenine, uridine or inosine. Alternatively, they mayinclude modified versions of any of these, such as those listed in Table1, or chemically modified nucleobases such as "protected" basesincluding, for example, a protecting group on the amino group of thenucleobase, such as, for example,

9-fluorenylmethylcarbonyl, benzyloxycarbonyl,

4-methoxyphenacyloxycarbonyl, t-butyloxycarbonyl,

1-adamantyloxycarbonyl, benzoyl, N-triphenylmethyl,

N-di-(4-methoxyphenyl) phenylmethyl.

FIG. 11. Examples of functional group manipulation in β-substitutedpyrroles as applied to the synthesis of rubyrins and rubyrin analogues.

FIG. 12. Examples of starting pyrroles for use in the synthesis ofrubyrins and rubyrin analogues.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Expanded porphyrins¹ are large pyrrole-containing macrocyclic analoguesof porphyrins (e.g. porphine, structure 1, FIG. 1), of which a numberare known¹⁻¹⁷. Prior to the present invention, only a few fullyconjugated examples had been reported that contain more than fourpyrrolic subunits, these are the smaragdyrins²,3 sapphyrins²⁻⁶pentaphyrins⁷,8 hexaphyrins⁹, and superphthalocyanines¹⁰, represented intheir generalized substituent-free forms as structures 2-6 (FIG. 1).

For the most part, the chemistry of the above-mentioned expandedporphyrin systems has been communicated in only the briefest fashion. Infact, at present, structural information is available only forderivatives of sapphyrin (e.g. 2)⁴,5 and pentaphyrin (e.g. 3)⁸ in theall-pyrrole series. In particular, no structurally characterizedhexapyrrolic macrocycles have previously been reported. Given thispaucity of information, the inventors considered that the preparationand study of new hexapyrrolic systems would be of interest. It wasreasoned that such studies would generate important information withregards to ring size, aromaticity, and effective macrocycle stability.

As mentioned above, it is known that the effectiveness of variousantiviral compounds, such as nucleotide analogues, is limited in vivodue to their inability to traverse lipophilic cell membranes²⁵,26. Infact, this has rendered many potential antiviral agents, including, forinstance, the anti-HIV agent, Xylo-G (FIG. 9d), largely or completelyinactive²⁷. A major aim of the present study was therefore to provide ameans of transporting active monophosphorylated forms of these putativedrugs (such as in FIG. 9e) into cells, which would allow a wide range ofotherwise inactive compounds to be used against viral infections.

Cystic fibrosis (CF) is the most common lethal genetic disease inCaucasians, striking about one in 2,500 U.S. infants⁶⁸. This disease,which is becoming increasingly well understood as the result of therecent identification of the responsible gene mutation, is characterizedby an inability to produce functional chloride anion channel proteins(the cystic fibrosis transmembrane regulator or CFTR protein)⁵⁵⁻⁵⁸ andby an inability to effect sufficient chloride and fluid excretion from,among others, pulmonary epithelial cells. This, in turn, leads to athick build up of mucous deposits in the lungs and to a higher thannormal susceptibility towards fatal pulmonary infections. It is theseinfections, often of the Pseudomonas aeruginosa type, that are generallythe causative agents of cystic fibrosis related death.

At present, the established treatment protocols for cystic fibrosisinvolve treating these secondary infections with appropriateantibiotics, as well as adjusting diet and removing by physical meansthe deleterious build up of mucociliary secretions⁵⁹. Unfortunately,these methods have not succeeded in prolonging the median lifeexpectancy of cystic fibrosis patients past the age of 25⁵⁹. Thus,considerable current effort is being devoted to developing treatmentsthat operate by attacking the underlying cause of disease. Here, avariety of approaches have been explored. These range from attempts atgene therapy (incorporating the normal, wild-type cystic fibrosis geneinto epithelia cells) to the administration of agents that restoreelectrolyte balance either by opening up other non-CFTR dependentchloride anion channels or by inhibiting cellular uptake of sodiumcations. Unfortunately, the viability of this latter electrolyte balancerestoration approach still remains limited.

In common with the problems associated with antiviral administration,the existence of a specific, in this case chloride-selective, anioncarrier could be of prime clinical utility in terms of treating a majorpublic health problem. It was the inventors aim in conducting thepresent study to develop a range of compounds that would allow both ofthese medically important anion transport problems to be addressed.

In defining these objectives, the biological importance of inter-linkedphosphate and halide transport was also considered. Transport of oneanionic species, achieved at the expense of the other, could conferconsiderable clinical advantage in the case of purely syntheticnucleotide carriers. It would allow one to achieve intracellulardelivery of an antiviral agent without creating a deleterious osmoticimbalance. Generally, for CF treatment, it is contemplated thatconcurrent out-of-cell chloride and sodium ion transport would be themost effective trigger for subsequent fluid excretion. However,simultaneous into-cell diffusion of, e.g., phosphorylated entities couldbe used to overcome problems associated with out-of-cell chloride aniontransport in the case of unfavorable chloride anion concentrationgradients.

Despite the previous preparation of several halide-binding receptors,none of these may be used as clinical chloride anion transporters. Forinstance, ammonium bicycles bind chloride anions with more specificitythan bromide and iodide⁶⁰, but only at low pH and with very low affinityconstants. Cryptand cations were found to bind halide anions with highaffinity and selectivity, and receptors with near-exclusive halidespecificities have been reported⁶¹,62. Unfortunately, these systemssuffer from a serious drawback as far as anion transport is concerned:They rely on a high net positive charge to effect the coordination of asingle anion. As a result, the supramolecular anion-to-receptor complexformed upon anion binding is still highly charged and relativelyinsoluble in organic media. Thus, through-membrane transport is onlyachieved if a large, organic soluble "helper anion" is added to thehalide-bearing transport medium⁶³.

This same problem of excess charge effects all the other knownpolyammonium halide anion receptors and all halide anion chelands of thepolyguanidinium type and pyridinium-based cyclophane class⁶⁴. Suchconsiderations of charge are of lesser concern in the case of the newermetal- and metalloid-derived anion receptors⁶⁵⁻⁶⁹. In this instance,however, questions of heavy metal toxicity cloud considerations ofpossible clinical utility. Thus, the need for good, neutralizingchloride anion carriers remains.

Regarding nucleoside transport, in preliminary work, the presentinventors employed triisopropylsilyl (TIPS) substituted (phosphate-free)nucleosides. It was found that efficient and selective through-membranetransport of non-charged nucleoside analogues could be achieved by usingthe complementary TIPS derivatives as carriers⁷⁰. Not surprisingly,however, these same TIPS derivatives proved completely ineffective astransport agents for the analogous phosphate-containing nucleotidederivatives. Thus, whilst confirming the viability of a base-pairingapproach to selective nucleotide recognition, this work served tohighlight further the need for an organic soluble, neutralizing,phosphate binding group.

In this light, the inventors reasoned that ditopic receptors capable ofrecognizing both the anionic phosphate and the neutral portions ofnucleotide derivatives, such as the purine or pyrimidine moieties, maybe particularly advantageous in the transport of anti-viral compounds.To synthesize such compounds, the inventors realized that they had toconsider the independent development of molecular recognition strategiesfor the complexation of two very different kinds of substrates (chargedanionic and neutral nucleobase) and then the subsequent co-combinationso as to provide a single receptor bearing both kinds of bindingsubunits. Further and significant problems to be overcome were thoseassociated with creating a molecule which has the capacity to bindanions and yet the ability to retain overall supramolecular chargeneutrality.

In initial studies using expanded porphyrins, the inventors determinedsapphyrin to be ineffective as a phosphate compound transporter, butpentaphyrin to be capable of transporting GMP at pH 6. Unfortunately,not only was this process found to be slow, but also extensivelyinhibited by the addition of chloride anion. In addition, sapphyrin andanthraphyrin^(18a),b were found to be capable of halide anion transport,but only in those pH regimes where they remain monoprotonated. Also, inboth cases the anion that was less well bound was found to betransported at the greatest rate (i.e. chloride by sapphyrin andfluoride by anthraphyrin)^(18a),b.

The search for an anion-binding compound with the ability to retainoverall supramolecular charge neutrality, led the inventors tosynthesize and characterize a novel class of hexapyrrolic expandedporphyrins, represented by structure 7 (FIG. 2), and substitutedderivatives thereof. Structure 7 corresponds to the macrocyclic system29,30,31,32,33,34-hexaazaheptacyclo[24.2.1.1²,5.1⁷,10.1¹²,15.1¹⁶,19.1²¹,24]tetratriaconta-1,3,5,7(31),8,10,12,14,16,18,20,22,24(34),25,27-pentadecaene(FIG. 2)¹⁹. Also contemplated by the invention are macrocyclic analoguesof structure 7, such as 8 and 9, which have the same overallconnectivity and same number of non-hydrogen atoms but which differ fromthe structurally characterized 26 π-electron prototypes by the number oftotal electrons contained within the π-electron periphery.

Substituted derivatives of compound 7 are red in dilute organicsolution, and the trivial name "rubyrin" (from the Latin rubeus) hastherefore been assigned to this new class of expanded porphyrins. Itwill be this trivial nomenclature that is used throughout the presentapplication. Thus, for instance,4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethyl-29,30,31,32,33,34-hexaazaheptacyclo[24.2.1.1²,5.1⁷,10.1¹²,15.1¹⁶,19.1²1,24]tetratriaconta-1,3,5,7(31),8,10,12,14,16,18,20,22,24(34),25,27-pentadecaene(structure 10a, FIG. 3), is named as4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethylrubyrin.

The rubyrin and rubyrin analogues of the present invention arecharacterized by the capacity to bind anions and yet the ability toretain overall supramolecular charge neutrality. A particular advantageto rubyrin molecules is their large size. This property renders rubyrinseasier to protonate than other macrocycles and makes them considerablymore effective at anion recognition and transport than any otherclasses, including the sapphyrins. The increased basicity of rubyrinrelative to sapphyrin is thus of considerable importance and representsa significant advance relative to the existing art.

The synthesis of the diprotonated form of4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethylrubyrin, structure17, contains two key steps. The first involves the acid catalyzedcondensation between the bis-α-free bipyrrole 11^(4a) and the acetoxyactivated pyrrole 12²⁰, to give the protected tetrapyrrolic derivative13. Standard near quantitative debenzylation then gave the diacid 14,which was used directly in an acid-catalyzed [4]+[2] MacDonald-type²¹oxidative condensation with the readily available^(4a) diformylbipyrrole, 15. The second step, condensation and air oxidation,ultimately provides the diprotonated rubyrin derivative 17a. Washingeither the purified ditosylate salt 17a, or the crude reaction mixture,with aqueous 10% hydrochloric acid then produced the more crystallinedihydrochloride derivative 17b. Carrying out a similar sequence startingwith diformyl bipyrrole 16²² gave in analogy salts 18a and 18b insimilar good yield.

Compounds 10a and 10b are synthesized from compounds 17 and 18. In bothcases, careful neutralization of dichloromethane solutions, achieved bywashing with aqueous sodium bicarbonate, was found to give rise to theneutral, free-base forms, corresponding to structures 10a and 10b (FIG.3) in the case of salts 17 and 18, respectively.

Additionally, several other rubyrin analogues are contemplated by thepresent invention. Different combinations of bipyrroles and pyrroles,for example see FIGS. 11 and 12, may be employed both in the first andsecond condensation steps to yield a variety of different macrocyclicproducts. The generation of further rubyrin analoques is particularlydescribed in Example VII, and represented in reaction schemes A throughE. A range of compounds with a wide variety of alkyl and/or arylsubstituents in the meso and/or β-pyrrolic positions, as exemplified bystructures 27, 35, 40 and 47, thus fall within the scope of the presentinvention.

Furthermore, rubyrin analogues employing heteroatoms are also within thescope of the present invention. In particular, different combinations ofoxygen and sulphur atoms, either alone, in combination with nitrogen, orin combination together, are contemplated. The synthesis of rubyrinanalogues in which two, four, or even six, of the nitrogen atoms havebeen replaced by oxygen or sulfur is described in Example X, andcompounds in accordance with those represented by structures 20-24 inFIG. 6 and structures 87, 92, 99 and 104 in reaction scheme P throughreaction scheme S, are therefore encompassed by the present invention.

The inventors contemplate that rubyrin itself and the unconjugatedrubyrin analogues of the present invention will be of use as drugdelivery agents. It is contemplated that they will find utility inmediating the cross-membrane transport of negatively charged compoundsor molecules, including halides, pseudohalides, such as azide or cyanideanions, or anionic clusters such as ferricyanide. The anion carryingproperties of rubyrins make them ideal candidates for the development ofsynthetic carriers capable of facilitating out-of-cell diffusion ofchloride anions, and hence for use as therapeutic agents for thetreatment of cystic fibrosis.

Importantly, rubyrin and analogues thereof are contemplated fortransporting phosphate-containing compounds into cells.Phosphate-containing compounds which may be transported in this mannerinclude, for example, simple alkyl or aryl phosphate, nucleotides suchas AMP or GMP, oligonucleotides and DNA or RNA, including anti-sense DNAor RNA constructs, and more particularly, antiviral compounds such asthose depicted in FIG. 9 (b,e,f,g,h,i), Table 1, and equivalentsthereof.

                  TABLE 1                                                         ______________________________________                                        MODIFIED NUCLEOSIDE/NUCLEOTIDE                                                ANALOGUE ANTI-METABOLITES                                                     ______________________________________                                        AraC            Erythrohydroxynonyladenine                                    AraAMP          Floxuridine                                                   Azaribine       Fluorouracil (5-FU)                                           Azathioprine    Idoxuridine                                                   Azauridine      COMPD                                                         AZT             Mercaptopurine                                                Bromodeoxyuridine                                                                             PFA                                                           Chlorodeoxyuridine                                                                            Thioguanine                                                   Cytarabine      Trifluoromethylde-oxyuridine                                  Deoxyuridine    Xylo-GMP                                                      DideoxyInosine DDI                                                            ______________________________________                                    

Any one of a variety of antiviral agents may be delivered to a cellusing rubyrin or a rubyrin analogue in accordance herewith. These agentsinclude, for example, the anti-HSV and anti-HIV agents acyclovirmonophosphate, Xylo-GMP, Ara-AMP, and/or phosphonate derivatives thatalso have documented anti-HSV and anti-HIV activity in vitro²⁸,29 (e.g.FIG. 9f and 9g), and simple species such as the pyrophosphatederivatives PFA (FIG. 9h) and COMDP (FIG. 9i) that have demonstratedanti-HIV reverse transcriptase activity in cell-free media³⁰.

As mentioned above, the large size, increased basicity and ease ofprotonation of rubyrins makes this class of macrocycles considerablymore effective at the recognition and transport of anions than otherclasses of molecules. The capacity of rubyrin, and analogues thereof, toeffect specific into-cell transport of anti-viral compounds iscontemplated to be of advantageous use against a wide variety ofdebilitating diseases, including, for example, herpes, hepatitis,measles, and AIDS. Such diseases are of major medical and economicimportance with AIDS being an international health problem and evenmeasles claiming over 100,000 lives per year world-wide²⁶.

Furthermore, the inventors reasoned that the rubyrin compounds of thepresent invention may be rendered even more useful as nucleotidetransporters if one or more nucleobase recognition units were to be"appended" directly onto the phosphate-chelating macrocyclic core. Thiswould impart a further degree of nucleotide specificity to binding andtransport reactions. Accordingly, rubyrin-nucleobase conjugates whichhave been derivatized by the addition of one or more nucleobasecompounds, as represented by the generalized structures IV and V (FIG.10), form an important aspect of the present invention. Rubyrinderivatives with one nucleobase per rubyrin molecule are referred to asditopic receptors, whereas those doubly-functionalized rubyrinderivatives with 2 nucleobases per molecule are termed tritopicreceptors.

Rubyrin mononucleobase derivatives may include any of thenaturally-occurring purine or pyrimidine nucleobases, namely, cytosine,guanine, thymidine, adenine, uridine or inosine. Equally, they mayinclude modified versions of any of these, such as the heterocycliccomponents of those nucleoside/nucleotide analogues listed in Table 1.Also included within the invention are the rubyrin mononucleobasederivatives including chemically modified nucleobase such as "protected"bases. Protecting groups are used to protect reactive groups, such asamino and carboxyl groups, from inappropriate chemical reactions.Rubyrin-nucleobase conjugates with protected bases include, for example,conjugates wherein one or more base has a protecting group, such as9-fluorenylmethylcarbonyl, benzyloxycarbonyl,4-methoxyphenacyloxycarbonyl, t-butyloxycarbonyl,1-adamantyloxycarbonyl, benzoyl, N-triphenylmethyl orN-di-(4-methoxyphenyl)phenylmethyl on the amino group of the nucleobase.

The present inventors contemplate many different chemical means by whichto connect nucleobases to rubyrin macrocycles. Various spacers may beused for the connection, such as, for example, oligomethylene bridgeswith terminal amino, or hydroxy function, which allow formation of amideand ester bond for the connection of the rubyrin and nucleobase units.This bridge may also be modified, e.g., by the reduction of the amidebond to give the amine function. Specific examples of the synthesis ofrubyrin-nucleobase conjugates are described in Example VIII and theresultant compounds are represented by structures 51, 55, 59 and 63 ofreaction schemes F through I.

Rubyrin nucleobase conjugates would be useful as antiviral adjuvants,capable of binding and solubilizing nucleotides and of effecting theirselective through-membrane transport at or near physiologic pH. Rubyrinnucleobase conjugates with appended oligonucleotides are alsocontemplated by the present invention, and would be of use in bindingand transporting oligo- or polynucleotides, including antisenseconstructs, into cells. As a mediator of DNA import, rubyrins mayconceivably be employed in the treatment of any disease in which thedelivery of an oligonucleotide or DNA fragment would be advantageous,such as in supplying a functioning gene, or in inhibiting an aberrantgene, for example, by employing an antisense DNA construct.

Another class of rubyrin derivatives or conjugates contemplated by thepresent inventors are the rubyrin saccharide derivatives which comprisea rubyrin macrocycle conjugated to a sugar, sugar derivative orpolysaccharide. The synthesis of rubyrin-saccharide compounds, asrepresented by structures 66, 68, 76 and 84 in reaction schemes J, K, Mand O, respectively, is described in Example IX. It will be understoodthat any one of a variety of individual sugar units, such as those setforth in Table 2, or polymers thereof, may be conjugated to rubyrin inaccordance herewith. Table 2 is intended to include modified versions ofthe sugar units, such as sugars having additional phosphate, methyl oramino groups and the like, and also includes D- and L-isomers and α andβ forms.

                  TABLE 2                                                         ______________________________________                                        Examples of Sugars and Sugar Derivatives                                      ______________________________________                                        Ribose           Fructose                                                     Arabinose        Sorbose                                                      Xylose           Tagatose                                                     Lyxose           Fucose                                                       Allose                                                                        Altrose          Methylglucoside                                              Glucose          Glucose 6-phosphate                                          Mannose                                                                       Gulose           N-Acetylgalactosamine                                        Idose            N-Acetylglucosamine                                          Galactose        Sialic Acid                                                  Talose                                                                        Ribulose                                                                      Xylulose                                                                      Psicose                                                                       ______________________________________                                    

In addition to the rubyrin analogues described above and the nucleobaseand saccharide conjugates, it will be appreciated that a variety ofother substituents, of desirable chemical function, may be appended to afunctionalized rubyrin moiety to create a rubyrin-based conjugate. Thepresent inventors contemplate the synthesis of rubyrin conjugatesincluding, for example: metal chelator moieties such as EDTA, EGTA,1,10-phenanthralene, DTPA, DOTA, crown ether, azacrown, catecholate andethylene diamine; alkylating agents such as ethylene diamine, epoxideand bromoacetamide; steroids and steroid derivatives; amino acids,peptides and polypeptides; other rubyrins, rubyrin derivatives,polymeric rubyrin, or other macrocyclic compounds such as sapphyrins,texaphyrins or derivatives thereof; and polymeric matrices or solidsupports such as polymers, glasses, agarose, polyacrylamide, controlledpore glass, silica gel, polystyrene and sepharose.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE I SYNTHESIS OF RUBYRIN STRUCTURES 17a and 17b

The synthesis of the diprotonated form of4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethylrubyrin, structure17, is shown in FIG. 4. It contains two key steps. The first involvesthe acid catalyzed condensation between the bis-α-free bipyrrole 11⁴ aand the acetoxy activated pyrrole 12.²⁰. This reaction, which wascarried out in analogy to earlier syntheses of symmetrictripyrranes^(16d), gave the protected tetrapyrrolic derivative 13 inapproximately 66% yield.

In more detail, compounds 11 (3.72 g, 17.2 mmol) and 12 (10.9 g, 34.5mmol) were combined in a round bottom flask with absolute isopropylalcohol (140 ml) and the resulting mixture heated at 80° C. to effectcomplete dissolution. p-Toluenesulfonic acid monohydrate (30 mg) wasthen added and the resulting green solution heated at reflux for 10hours under nitrogen. The resulting suspension was then cooled to roomtemperature and placed in a refrigerator for one hour. Filtration,washing with cold ethanol, and drying in vacuo then afforded compound 13as a fine, off-white powder (8.24 g, 66%), which could be furtherpurified by recrystallization from CH₂ Cl₂ /MeOH. m.p.=163°-166° C.; ¹ HNMR (300 MHz, CDCl₃): δ0.83 (t, 6H, CH₂ CH₃), 1.10 (t, 6H, CH₂ CH₃),2.02 (s, 10H, CH₃ and CH₂ CH₃), 2.12 (s, 6H, CH₃), 2.46 (m, 4H, CH₂CH₃), 3.70 (bs, 6H, pyrrole-CH₂ and CH₂ Ph), 4.55 (bs, 2H, CH₂ Ph), 6.85(m, 4H, aromatic), 7.21 (m, 6H, aromatic), 9.89 (bs, 2H, NH), 10.98 (bs,2H, NH); ¹³ C NMR (75.5 MHz, CDCl₃): δ=11.20, 11.30, 15.21, 15.61,16.86, 18.20, 22.34, 65.91, 113.97, 117.12, 120.76, 121.29, 123.36,123.51, 126.53, 126.88, 127.64, 127.82, 133.80, 135.97, 164.48; HRMS(CI): m/z 726.4142 (M⁺) calcd for C₄₆ H₅₄ N₄ O₄ 726.4145 (±0.0003).

Standard near quantitative debenzylation of 13 then gave the diacid 14.This was achieved by dissolving the dibenzyl ester 13 (0.24 g, 0.33mmol) in dry THF (100 ml) and subjecting to hydrogenation over 10%palladium-charcoal (40 mg) at 1 atm H₂. The catalyst was then separatedby filtration, the solvent reduced in volume on a rotary evaporator, andthe product precipitated by trituration with hexanes. The pale blueprecipitate was collected by filtration, dried in vacuo (to yield 0.16 g(90%) of 18).

The diacid, 14, was used directly, without delay, in the next step, thesynthesis of 17a. This reaction is an acid-catalyzed [4]+[2]MacDonald-type²¹ oxidative condensation with the readily available^(4a)diformyl bipyrrole, 15. This condensation and the accompanying airoxidation, which taken together represent the second critical step inthe overall synthetic sequence, then provide, following work-up andchromatographic purification, the diprotonated rubyrin derivative, 17ain roughly 20% yield. Washing either this purified ditosylate salt orthe crude reaction mixture with aqueous 10% hydrochloric acid thenproduced the more crystalline dihydrochloride derivative, 17b in yieldsof 63% (based on 17a) and 40% (based on 14), respectively.

In more detail, to create 17a, 17b, and 19, compounds 15 (136 mg, 0.5mmol) and 14 (273 mg, 0.5 mmol) were dissolved with warming in 1.0 L ofabsolute ethanol, the mixture allowed to cool to room temperature and2.0 g of p-toluenesulfonic acid monohydrate added all at once. Oxygenwas then bubbled through the stirred mixture for 18 hours. The ethanolwas then removed on a rotary evaporator and the dark purple residuetaken up in CHCl₃ and purified by column chromatography (Merck type 60(230-400 mesh) Silica gel) using first CHCl₃ and then CHCl₃ /MeOH(98/2)as the eluents. Collection of the dark red fraction and removal of thesolvent gave 105 mg (20%) of 17a which could be purified further byrecrystallizing from CHCl₃ /hexanes. Collection of the violet fractionalso yielded small (≦10% yield) amounts of 19.

Dissolving compound 17a in dichloromethane and washing with 10% HCl for2 hours at room temperature then afforded, after work up, product 17b inca. 63% yield. This same material could also be obtained in roughly 40%net yield by treating the crude condensation product (from 14 and 15)with 10% HCl prior to work up and chromatography. Single crystals of 17bwere obtained by vapor diffusion recrystallization from CHCl₃ /pentane.

For 17a: decomp. above 220° C.; ¹ H NMR (300 MHz, CDCl3): δ=-4.14 (s,2H, NH), -3.68 (s, 4H, NH), 1.50 (s, 6H, PhCH₃), 1.90 (d, J_(HH) =7 HZ,4H, phenyl H), 2.30 (m, 18H, CH₂ CH₃), 3.90 (s, 12H, CH₃), 4.10 (m, 8H,CH₂ CH₃), 4.30 (s, 6H, CH₃), 4.70 (q, 4H, CH₂ CH₃), 4.90 (d, J_(HH) =7Hz, 4H, phenyl H), 11.18 (m, 4H, meso-H); HRMS (FAB): m/z 694.4707 (M⁺-2TsO⁻), calcd for C₄₆ H₅₈ N₆ 694.4723 (±0.0016).

For 17b: decomp. above 230° C.; ¹ H NMR (300 MHz, CDCl₃): δ=-5.30 (s,2H, NH), -4.97 (s, 4H, NH), 2.09 (m, 12H, CH₂ CH₃), 2.34 (t, J_(HH) =7Hz, 6H, CH₂ CH₃), 4,06 (s, 12H, CH₃), 4.40 (s, 6H, CH₃), 4.43 (m, 8H,CH₂ CH₃), 4.82 (q, J_(HH) =7 Hz, 4H, CH₂ CH₃), 11.58 (s, 2H, meso-H)11.60 (s, 2H, meso-H); ¹³ C NMR (75.5 MHz, CDCl₃): δ=12.98, 15.73,17.50, 17.99, 21.05, 21.29, 92.74, 92.88, 126.47, 127.30, 129.03,129.65, 130.73, 136.38, 139.32, 143.31; UV/VIS (CH₂ Cl₂): λ_(max) [nm](ε)=505 (302,000), 711 (11,000), 791 (15,500), 850 (38,000); HRMS (FAB):m/z 694.4660 (M⁺ -Cl₂), calcd for C₄₆ H₅₈ N₆ 694.4723 (±0.0063).

For 23: ¹ H NMR (300 MHz, CDCl₃): δ=0.98 (m, 9H, CH₂ CH₃), 1.09 (m, 9H,CH₂ CH₃), 2.06 (m, 18H, CH₃), 2.37 (m, 6H, CH₂ CH₃), 2.57 (m, 6H, CH₂CH₃), 6.78 (m, 4H, "meso"-H), 11.85 (s, 1H, NH), 12.32 (s, 1H, NH),12.51 (s, 1H, NH), 12.60 (s, 1H, NH), 12.62 (s, 1H, NH), 12.74 (s, 1H,NH); MS (FAB): m/z 696 (M⁺ +1, 4%), 695 (M⁺, 5%), 307 (22%), 154 (100%);UV/VIS (CH₂ Cl₂): λ_(max) =538 nm.

EXAMPLE II SYNTHESIS OF RUBYRIN STRUCTURES 18a and 18b

Employing the same rubyrin synthetic methodology as described in ExampleI, but substituting the starting compounds for different startingmaterials allows a wide range of other rubyrin analogues to be prepared.For example, starting with diformyl bipyrrole 16²² and performing thesynthesis in the same manner as described above, gave in analogy salts18a and 18b in similar good yield. From condensation of 14 (1.1 g, 2mmol) and 16 (0.545 g, 2 mmol), 220 mg of compound 18a was obtained(16%).

¹ H NMR (300 MHz, CDCl₃): δ=-5.66 (s, 4H, NH), -5.40 (s, 2H, NH), 1.75(t, J_(HH) =7, 6H, CH₂ CH₂ CH₃), 2.13 (t, J_(HH) =7, 6H, CH₂ CH₃), 2.36(t, J_(HH) =7, 6H, CH₂ CH₃), 3.06 (m, 4H, CH₂ CH₂ CH₃), 4.24 (s, 6H,CH₃), 4.51 (s, 6H, CH₃), 4.77 (q, J_(HH) =7, 4H, CH₂ CH₃), 5.06 (q,J_(HH) =7, 4H, CH₂ CH₃), 11.17 (s, 2H, "meso"-H), 12.00 (s, 2H,"meso"-H), 12.29 (s, 2H, b-H); MS (FAB): m/z 694 (M⁺ -2TsO⁻); UV/VIS(CH₂ Cl₂) λ_(max) =501 nm.

EXAMPLE III CHARACTERIZATION OF RUBYRIN STRUCTURES 17 and 18

Formally, rubyrin 10 and its derivatives may be considered as 26π-electron annulenes. Thus, the diprotonated salts 17 and 18 wereexpected to be aromatic. The available spectroscopic data is consistentwith this aromatic formulation. For instance, low-field meso-likemethine signals and high-field internal NH resonances were observed inthe ¹ H NMR spectra as would be expected for a large aromatic expandedporphyrin system. In the specific case of 17b in CDCl₃ these signalswere observed at 11.58 and 11.60 ppm (in a 1:1) ratio, and at -4.97 and-5.30 ppm (in a 2:1 ratio), respectively, values which compare closelyto those observed for the dihydrochloride salt of3,8,12,13,17,22-hexaethyl-2,7,18,23-tetramethylsapphyrin [H₂.Sap]²⁺measured under the same experimental conditions^(4a).

Additionally, both 17 and 18 display Soret-like and Q-type transitionsin their optical spectra, which, as would be expected for a largearomatic expanded porphyrin, are considerably red-shifted as compared tothose of smaller porphyrin-like systems. In fact, relative to [H₂.Sap]²⁺and [H₂.OEP]²⁺ (the diprotonated derivative of2,3,7,8,12,13,17,18-octaethylporphyrin) measured under similarexperimental conditions, the Sorer band of 17b is shifted by ca. 50 and100 nm, respectively (c.f. FIG. 7). In the case of the lowest energyQ-type band, the corresponding shifts are on the order of 180 and 270nm, respectively.

Further evidence for the aromatic nature of rubyrin was obtained fromthe X-ray diffraction structure of 17b. The experimental details are asfollows: (C₄₆ H₅₆ N₆ H₂)²⁺ (Cl⁻)₂ (CHCl₃)₂ ; triclinic, P1 (No. 2), Z=1in a cell of dimensions: a=10.196(8), b=11.141(8), c=13.476(9) Å,α=67.18(5), β=72.97(6), γ=68.56(6)°, V=1293(2) Å³, ρ_(calc) =1.29 g cm⁻³(173K), F(000)=526. Data collected at 173K on a Nicolet R3diffractometer, graphite monochromatized Mo _(K)α radiation (λ=0.7107 Å)using the Ω-scan technique out to 50° in 2θ; 4573 unique reflections,2871 with F_(o) ² >3σ(F_(o) ²) The structure was solved by directmethods and refined by full-matrix least-squares (SHELXTL-Plus, NicoletXRD, Madison, Wis., USA) with anisotropic thermal parameters for thenon-hydrogen atoms. Hydrogen atoms on C18, C19, C19A, C20, C21, C22,C22A and C23 were idealized (C--H 0.96Å) while all others were takenfrom a ΔF map. All were refined isotropically. The complex lies aroundan inversion center at 0,1/2,1/2 resulting in the occupational disorderof the ethyl and methyl groups of the pyrrole ring containing N2. TheCl⁻ ions are H-bonded to the pyrrolic hydrogens and to the CHCl₃ solvatemolecules. The final R=0.0529, R_(w) =0.0678, goodness of fit=2.284 for361 parameters. The minimum and maximum peaks in the final ΔF map were-0.30, 0.38 e³¹ Å⁻³ respectively

As illustrated in FIG. 8, X-ray diffraction studies revealed structure17 to have a near planar conformation for the core macrocycle that isonly slightly waffled by virtue of interactions with the hydrogen-boundchloride counter anions (the maximum deviation (C2) of the C and Nmacrocycle atoms from the mean plane is 0.509(4) Å). In fact, this lackof distortion is reminiscent of that seen in the X-ray structure of [H₂.OEP]²⁺ (Ref. 23) and several recently-reported diprotonatedbisvinylogous expanded porphyrins^(13b),13c, suggesting that significantthrough-cycle π-electron conjugation pertains in 17b. Consistent withthis conclusion is the finding that the average carbon-carbon andcarbon-nitrogen bond distances within the formal 26 π-electron periphery(1.394 Å and 1.375 Å, respectively) are close in value to those observedin the corresponding diprotonated OEP structure (1.390 Å and 1.375 Å,respectively)^(23a).

Interestingly, however, the inter-pyrrole angles about the bridgingmethines (137.0° and 137.6°) are considerably greater than those foundin protonated porphyrins (wherein the in-core angles are roughly127°)²³. Thus, the expanded porphyrin 17b displays an inner core that ismore open than might perhaps be expected for what may formally beconsidered as two directly appended "three-quarter porphyrins" linkedvia two sets of bipyrrole-defining bonds. Nonetheless, it is clear thatfrom a structural point of view, compound 17b displays all that isexpected of an aromatic system.

EXAMPLE IV SYNTHESIS OF RUBYRIN STRUCTURES 10a and 10b

The synthesis of structure 10a,4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethyl-29,30,31,32,33,34-hexaazaheptacyclo[24.2.1.1²,5.1⁷,10.1¹²,15.1¹⁶,19.1²¹,24]tetratriaconta-1,3,5,7(31),8,10,12,14,16,18,20,22,24(34),25,27-pentadecaene (hereintermed 4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethylrubyrin) wasachieved as described below.

To synthesize compounds with structures 10a and 10b, the synthesis ofcompounds 17 and 18 was first conducted, as described above in ExampleIII. In both cases, careful neutralization of dichloromethane solutions,achieved by washing with aqueous sodium bicarbonate, was found to giverise to the neutral, free-base forms, corresponding to structures 10aand 10b (FIG. 3) in the case of salts 17 and 18, respectively.

Specifically, this neutralization is carried out by dissolving salt 17or 18 in CH₂ Cl₂ in a one-neck round bottomed flask and adding saturatedaqueous NaHCO₃. The two-layer mixture is then stirred via a magneticstirring apparatus for two hours. Separating the organic layer from theaqueous layer, and washing the organic layer with de-ionized water inthe same fashion described above, drying the organic layer overanhydrous sodium sulfate and removing the solvent on a rotary evaporatoraffords the neutral, free-base rubyrin analogue 10a or 10b.

Interestingly, the free-base form of 10b was generally found to be morestable. However, in both cases, the free-base form proved ratherunstable. Thus, as described herein, full characterization was effectedusing the diprotonated forms, 17 and 18.

EXAMPLE V SYNTHESIS OF RUBYRIN ANALOGUE STRUCTURE 19

In addition to those described above, several other products wereobtained in the course of the above condensations. In the case ofprimary product 17a, an effort was made to characterize these materialsand two such minor products were thus identified. The first of these wasfound to be a sapphyrin derivative, namely3,7,12,18,22-pentaethyl-2,8,13,17,23-pentamethylsapphyrin.

The second was a partially reduced, rubyrin-like system,4,8,13,18,23,27-hexaethyl-3,9,14,17,22,28-hexamethyl-29,30,31,32,33,34-hexaazaheptacyclo[24.2.1.1²,5.1⁷,10.1¹²,15.1¹⁶,19.1²¹,24]tetratriaconta-1,3,5,7,9,11,13,15,17,19,21,23,25,27-tetradecaene(structure 19, FIG. 5). This compound (structure 19) was received as abyproduct of the reaction sequence to produce compound 17a. It has thesame connectivity but is of different oxidation state. Nonetheless, thismaterial appeared to be stable to the reaction conditions. Still,however, it could be converted to 17b in near quantitative yield bytreating with DDQ in the presence of acetic acid and then washing with10% HCl. Thus, at the present time, it is not clear whether compound 19itself (as opposed to some other reduced rubyrin species) functions asthe actual intermediate under the present condensation conditions.

In any case, this well characterized product, compound 19, which isbright violet in color, stands as a specific embodiment of compounds ofthe general class represented by structure 8 (FIG. 2). Its existence andisolation thus stands as evidence inter alia that compounds with thesame connectivity and total non-hydrogen atom count as rubyrin, asexemplified by structure 8, can be prepared according to the methods ofthe present invention.

EXAMPLE VI SYNTHESIS OF RUBYRIN ANALOGUES REPRESENTED BY STRUCTURE 9

In addition to those compounds, such as 19, represented by structure 8(FIG. 2), compounds based upon structure 9 will also have the sameconnectivity and total non-hydrogen atom count as rubyrin. Thesecompounds may also be prepared according to the synthetic methodologydisclosed herein. For example, the hexamethyl hexaethyl derivative of 9may be an as-yet uncharacterized byproduct of the reaction used toprepare 17. Alternatively, it is contemplated that this material may beobtained by controlled reduction of 17 using hydride reagents and/orcontrolled hydrogenation.

EXAMPLE VII SYNTHESIS OF SUBSTITUTED RUBYRINS AND FURTHER ANALOGUES

It will also be apparent to one of skill in the art of organic chemistrythat the two rubyrins presented in FIG. 4 (or their reduced congeners asrepresented by compounds such as 19) are not the only ones that can beobtained within the context of this synthetic methodology. For example,firstly, the inventors contemplate that the choice of bipyrroles for usein the second condensation step, as illustrated by the use of 15 and 16(to make 17 and 18), may be varied. Secondly, it is also envisioned thatdifferent combinations of bipyrroles and pyrroles may be employed in thefirst condensation step to produce various analogues of 13 and 14.

A preliminary example concerns the inventors' use of5,5'-diformyl-4,4'-dipropyl-2,2'-bipyrrole as a starting material foruse in reaction sequences in accordance with the present invention. Thissynthetic process involved reaction with compound 12 followed bydebenzylation and subsequent condensation with compound 16 to yield ananalogue of compound 10,8,23-diethyl-4,13,18,27-tetrapropyl-9,22-dimethylrubyrin, in which fourof the 12 possible β-pyrrolic positions are unsubstituted by alkylgroups. Satisfactory NMR and high resolution mass spectrometric data wasobtained for this product.

Accordingly, it is contemplated that a variety of approaches may beemployed, in accordance with the present invention, to prepare systemsbearing a wide variety of alkyl and/or aryl substituents in the mesoand/or β-pyrrolic positions.

For example, meso substituted compounds may be prepared to test theextent to which the presence of different groups, such as, for example,4'-phenyl and/or 2'-phenyl donating groups can be made to augmenteffective nitrogen lone pair basicity and/or enhance higher pH phosphatetransport capability. Extensions to systems bearing two (or more) mesosubstituents are also contemplated within the scope of the invention. Inany event, it is important to appreciate that by adding further mesosubstituents one will, in all likelihood, induce substantial distortionsof the macrocycle off planarity. Thus, these syntheses should providecompounds that will allow steric effects to be assessed. Naturally, itis contemplated that transport and pK_(a) ' tests will be conducted todetermine the effects of the various substitutions.

In preliminary work, to date, several phenyl-bearing rubyrins have beenprepared by a "direct insertion" procedure. This procedure (a specificexample of which is shown in Reaction Scheme A), which is necessarilyinefficient, involves the condensation of a bis-α-free bipyrrole (e.g.,25) with an α, ω-free bis(pyrrolyl)-bipyrrole (e.g., 26) in the presenceof benzaldehyde or substituted benzaldehyde under rubyrin formingconditions, as described herein, to afford bisarylrubyrin 27. Thisinefficiency reflects the fact that in addition to the bisarylrubyrinproducts, one also obtains a range of other macrocyclic andnon-macrocyclic products.

Improved syntheses can be envisioned, however, in which an α-free,ω-protected bipyrrole (e.g., structure 28 in Reaction Scheme B), isco-condensed with an α-free pyrrole (e.g., 29) in the presence of anaromatic aldehyde, as in Reaction Scheme B. The benzyl ester of theresulting pyrrolyl-bipyrrole can then be selectively cleaved understandard debenzylation conditions and decarboxylated withtrifluoroacetic acid to give the α-free pyrrolyl-bipyrrole, structure30. Acid catalyzed condensation of pyrrolyl-bipyrrole 31 with anothermolecule of α-free pyrrole 28 in the presence of an aromatic aldehydeyields an ethyl ester-protected diaryl, bis(pyrrolyl)-bipyrrole 32,which can be deprotected to form the α, ω-free bis(pyrrolyl)-bipyrrole33. Acid catalyzed co-condensation of the tetrapyrrolic fragment 33 withan α, ω-free bipyrrole (e.g., 34) in the presence of an aromaticaldehyde, under rubyrin-forming conditions such as those described inExample 1, affords a rubyrin with each of its four "meso" positionssubstituted with aryl groups (e.g., 35, Reaction Scheme B). ##STR1##

It will, of course, be understood that in syntheses such as thoseexemplified in Reaction Scheme B, substituents in the startingcompounds, such as those groups represented by R¹, R², R³ and R⁴, may bevaried as desired. Such groups may separately and independently include,for example, H, alkyl, aryl, amino, hydroxyl, alkoxy, carboxy,carboxamide, ester, amide, sulfonato, hydroxy substituted alkyl, alkoxylsubstituted alkyl, carboxy substituted alkyl, amino substituted alkyl,sulfonato substituted alkyl, ester substituted alkyl, amide substitutedalkyl, substituted aryl, substituted alkyl, substituted ester,substituted ether, or substituted amide groups. Thus, by using theappropriately substituted starting units, a rubyrin product may beproduced in which groups R₁ -R⁶ (as represented by structure 35) may besubstituted with any of the groups listed above.

Extensions of known methods can also be straightforwardly conceived thatwould allow one of skill in the art, in light of the present disclosure,to prepare functionalized rubyrins bearing one or more non-alkylsubstituents in the β positions. As is true for the sapphyrins^(4a),80the easiest entry into such systems involves the preparation of carboxyalkyl substituted systems, for example, compounds bearing substituentssuch as --(CH₂)_(n) --CO₂ H, and then further elaborating these to formthe corresponding hydroxy alkyl, amino alkyl, thiol, sulfanato, ether,amide, ester, or formyl alkyl derivatives.

In addition to the direct conversions described above, it is importantto appreciate that carboxy alkyl substituted rubyrins can also be usedas the basis for obtaining other, more complex functionalized systems.For instance, as described by Kral et al, 1992⁷⁷ incorporated herein byreference, for the sapphyrin series, the carboxylic acid group of thecarboxy alkyl bearing rubyrins can be activated using standard reagents(such as, for example, thionyl chloride or DCC) and used to prepareeither ester- or amide-linked conjugates.

Again, in analogy to the sapphyrins, said conjugates could includecompounds that contain one or more nucleic acid base ("nucleobase") orsugar ("saccharide") subunits, as is described in detail in thefollowing Examples (Examples VIII and IX). For example, in suchsyntheses, one may condense a protected amino alkyl nucleobase, forinstance, the known material1-(2-aminoethyl)-4-[triphenylmethyl)amino]-pyrimidin-2-one⁷⁸, with theactivated rubyrin carboxylic acid and subsequently effect nucleobasedeprotection. Similarly, one may employ an amino-bearing protectedsugar, such as per-O-acetylated glucosamine, and subsequentdeprotection. In addition, one can use such a conjugation approach toprepare complex ethers, esters, or amides where the ether, ester, oramide linkages are used to append a wide variety of polyfunctionalsubstituted alcohol and/or amine fragments on to the rubyrin periphery.

The requisite carboxy alkyl substituted rubyrins can be prepared byseveral routes. Two of these bear direct analogy to the earlier work inthe sapphyrin series and thus are chosen for highlight in this example.First, in analogy to the preparation of3,12,13,22-tetraethyl-8,17-bis(carboxyethyl)-2,7,18,23-tetramethylsapphyrin^(4a),condensation of benzyl5-acetoxymethyl-3-methyl-4-(methoxycarbonylethyl)-pyrrole-2-carboxylate,structure 37, with bipyrrole 36 can be used to obtain, followingdebenzylation,bis(5-carboxy-3-(methoxycarbonylethyl)-4-methyl-pyrrol-2-ylmethyl)-2,2'-bipyrrole38b (Reaction Scheme C). This compound, following condensation withbipyrrole 39, under rubyrin-forming conditions such as those describedin Example 1, will provide the rubyrin system 40 bearing two carboxyalkyl substituents (Reaction Scheme C). In the structures represented inscheme C, R¹, R², R⁴, and R⁵, in the starting units and the product, mayseparately and independently include H, alkyl, aryl, amino, hydroxyl,alkoxy, carboxy, carboxamide, ester, amide, sulfonato, hydroxysubstituted alkyl, alkoxyl substituted alkyl, carboxy substituted alkyl,amino substituted alkyl, sulfonato substituted alkyl, ester substitutedalkyl, amide substituted alkyl, substituted aryl, substituted alkyl,substituted ester, substituted ether, or substituted amide groups.

Second, in retrosynthetic analogy to the more recent preparation of3,8,17,22-tetraethyl-12-(carboxyethyl)-2,7,13,18,23-pentamethylsapphyrin.sup.77,benzyl 3,5-dimethyl-4-(methoxycarbonylethyl)-pyrrole-2-carboxylate 41may be converted, as shown in Reaction Scheme D, to its correspondingbipyrrole 44a(3,3'-bis(methoxycarbonylethyl)-5,5'-bis(benzyloxycarbonyl)-4,4'-dimethyl-2,2'-bipyrrole)via sulfuryl chloride oxidation to acid 42, followed by standardiodination (43), and copper bronze mediated Ullman coupling^(4a), 82.Following standard debenzylation to produce 44b and Clezy formylationwith trifluoroacetic acid (TFA) and triethylorthoformate, condensationof the resulting bipyrrole 45(3,3'-bis(methoxycarbonylethyl)-5,5'-diformyl-4,4'-dimethyl-2,2'-bipyrrole)with the tetrapyrrolic fragment 46 of Reaction Scheme E, under normalrubyrin-forming conditions such as those described in Example I, willprovide a rubyrin containing at least two carboxy alkyl substituents, atpositions 4 and 27, protected as their corresponding methyl esters(structure 47, Reaction Scheme E). As with other examples presentedherein, R¹, R², and R³ of structures 46 and 47 may separately andindependently include H, alkyl, aryl, amino, hydroxyl, alkoxy, carboxy,carboxamide, ester, amide, sulfonato, hydroxy substituted alkyl, alkoxylsubstituted alkyl, carboxy substituted alkyl, amino substituted alkyl,sulfonato substituted alkyl, ester substituted alkyl, amide substitutedalkyl, substituted aryl, substituted alkyl, substituted ester,substituted ether, or substituted amide groups. ##STR2##

EXAMPLE VIII SYNTHESIS OF RUBYRIN-NUCLEOBASE CONJUGATES

It is contemplated that suitable rubyrin-based dibasic phosphatechelators may be modified so as to obtain ditopic binding systems thatdisplay high inherent specificity for a given purine or pyrimidinederived nucleotide. This may be achieved by adding a synthetic appendageof a nucleobase moiety. For example, the known aminobutyl cytosinederivative may be employed with an acid-catalyzed detritylationprocedure.

Alternatively, the mode of base attachment may be varied, for example,at the level of coupling, protecting group, precursors, and the length,nature, and orientation of any linking groups. The effects of suchvariations on yield, selective binding, and other properties of theresultant rubyrin-based molecule, such as, for example, effectivethrough-membrane transport capability may then be determined and anyadjustments made accordingly. For example, the nature of R groups may bechanged, or 2° amides replaced by 3° ones

In principle, based on preliminary studies which demonstrated thefeasibility of achieving nucleobase "chelation" via complementaryWatson-Crick type base-pairing interactions, these compounds shoulddisplay base-selective transport capability. This may be specificallyexamined by various structural, static binding, and dynamic transportanalyses. In particular, it will be determined whether thecytosine-for-guanine selectivity that the inventors observed in mixed(i.e. non-covalently linked) systems²⁴ holds in the case of suitablydesigned synthetic conjugates. In addition, it will be tested whetherthis same base-pairing approach suffices to engender nucleobaseselectivity in the case of adenine-thymine pairing.

As an extension of the above analyses, doubly functionalized systems maybe synthesized for use in the selective binding of dinucleotides, someof which have interesting antiviral properties⁷², as well as for therecognition and transport of mononucleotides. In the latter case, it issuggested, the possible combination of both Watson-Crick and Hoogsteentype base-pairing interactions could confer a degree of specificity notavailable using simpler systems. To the extent this proves true, it ispossible that the doubly functionalized rubyrin system could serve as aviable antiviral adjuvant, capable not only of binding and solubilizingthe phosphate portion of a nucleotide monophosphate, but also ofeffecting its selective through-membrane transport at or nearphysiologic pH.

The studies carried out in the sapphyrin series stands as an allegory ofsuccess. For instance, it was found that the attachment of a nucleobaseto a sapphyrin core greatly enhanced the nucleotide recognitionselectivity for transport. Thus the inventors expect that thefunctionalization of rubyrins, which because of their larger core sizeand increased basicity are inherently much better for anion recognitionand phosphate anion chelation than sapphyrins, will lead to systems oftremendous superiority relative to any produced to date.

Reaction Schemes F through I represent examples of the synthesis ofrubyrin nucleobase conjugates. The synthetic methodology represented inthese reaction schemes may be straightforwardly adapted for thesynthesis of any given rubyrin nucleobase compound by employing thedesired starting materials. Groups R¹, R², R⁴, and R⁵ in ReactionSchemes F and G and groups R¹, R² and R³ in Reaction Schemes H and I,may separately and independently include H, alkyl, aryl, amino,hydroxyl, alkoxy, carboxy, carboxamide, ester, amide, sulfonato, hydroxysubstituted alkyl, alkoxyl substituted alkyl, carboxy substituted alkyl,amino substituted alkyl, sulfonato substituted alkyl, ester substitutedalkyl, amide substituted alkyl, substituted aryl, substituted alkyl,substituted ester, substituted ether, or substituted amide groups.

The first example of the preparation of a rubyrin nucleobase conjugateis shown in Reaction Scheme F. Here, a dicarboxy alkyl bearing rubyrin,the 8,23-bis(methoxycarbonylethyl)-rubyrin 48 can be saponified to itsdiacid form 49 by treatment with a 1:1 mixture of HCl andtrifluoroacetic acid. DCC coupling of rubyrin diacid 49 with tritylprotected aminoethyl cytosine 50 in methylene chloride at 0° C. followedby deprotection with TFA, as shown in Reaction Scheme F, affords theamide linked bis(aminoethyl)cytosine rubyrin conjugate 51.

Similarly, the amide linked bis(aminoethyl)guanosine rubyrin conjugate55 can be prepared by DDC coupling of rubyrin diacid 53 withbenzoyl-protected aminoethyl guanosie 54 in DMF at 0° C. followed bydeprotection with TFA as shown in Reaction Scheme G.

In another example of a dicarboxy alkyl bearing rubyrin, the4,27-bis(methoxycarbonylethyl)-rubyrin 56 (Reaction Scheme H) can besaponified to diacid 57 by treatment with a 1:1 mixture of HCl andtrifluoroacetic acid. DCC coupling of rubyrin diacid 57 withtrityl-protected aminoethyl cytosine 58 in methylene chloride at 0° C.followed by deprotection with TFA, as shown in Reaction Scheme H,affords the amide linked bis(aminoethyl)cytosine rubyrin conjugate 59.

The amide linked bis(aminoethyl)guanosine rubyrin conjugate 63 ofReaction Scheme I can be prepared by DDC coupling of rubyrin diacid 61with benzoyl-protected aminoethyl guanosine 62 in DMF at 0° C. followedby deprotection with TFA as shown in Reaction Scheme I. ##STR3##

EXAMPLE IX SYNTHESIS OF RUBYRIN-SACCHARIDE CONJUGATES

Rubyrin derivatives or conjugates including one or more saccharide unitsmay also be prepared according to the synthetic methodology describedhereinbelow. The sugar units (represented originally by structures 65and 75) in Reaction Schemes J through O are intended to represent anyindividual sugar or sugar derivative, such as those set forth in Table2, or polymers thereof, and include modified sugars, such as methyl,amino, and phosphate sugars, and D-, L-, α and β forms of said sugars.

The synthetic methodology represented in the following reaction schemesmay be straightforwardly adapted for the synthesis of any rubyrinsaccharide compound as desired. Groups R¹, R², R⁴, and R⁵ in ReactionSchemes J, L and M, and groups R¹, R² and R³ in Reaction Schemes K, Nand O, may separately and independently include H, alkyl, aryl, amino,hydroxyl, alkoxy, carboxy, carboxamide, ester, amide, sulfonato, hydroxysubstituted alkyl, alkoxyl substituted alkyl, carboxy substituted alkyl,amino substituted alkyl, sulfonato substituted alkyl, ester substitutedalkyl, amide substituted alkyl, substituted aryl, substituted alkyl,substituted ester, substituted ether, or substituted amide groups.

For the preparation of amide-linked rubyrin saccharide conjugates, suchas those represented by structure 66, diacid chloride substitutedrubyrin 64 (Reaction Scheme J) may be prepared by treating itsrespective diacid rubyrin, formed as shown in Reaction Scheme J, withthionyl chloride. The rubyrin diacid chloride 64 thus prepared can becoupled with the acetoxy protected HBr salt of amino saccharide 65 inmethylene chloride and pyridine, and deprotected with KOH in methanol asin Reaction Scheme J.

In the same fashion, the 4,17-bis(acid chloride)rubyrin 67 (ReactionScheme K) can be prepared by treating its respective diacid rubyrin,formed as shown in Reaction Scheme H, with thionyl chloride. Theamide-linked bis(saccharide) rubyrin 68b may be prepared by couplingrubyrin diacid chloride 67 with the acetoxy protected HBr salt of aminosaccharide 65 in methylene chloride and pyridine, followed bydeprotection with KOH in methanol as illustrated in Reaction Scheme K.

To prepare ether-linked bis(saccharide)rubyrin conjugates, the dihydroxyfunctionalized rubyrins, such as those represented by structure 73b inReaction Scheme L, may be prepared. For example, reduction of the methylesters of tetracycle 69 with borane-THF to the corresponding alcoholsfollowed by protection with acetic anhydride provides theacetoxy-protected diol 71a. Standard debenzylation to afford 71bfollowed by condensation with diformyl bipyrrole 72, underrubyrin-forming conditions such as those described in Example I,provides the acetoxy-protected dihydroxy rubyrin 73a. Deprotection ofthe hydroxyl groups can be achieved by treatment with HCl in methanol toafford dihydroxyrubyrin 73b.

Dihydroxyrubyrins, formed as described above (Reaction Scheme L), can becoupled with acetoxy- and/or benzoyl-protected bromo-substitutedsaccharide units, such as structure 75 (Reaction Scheme M), in methylenechloride with silver triflate and barium carbonate. This results in theproduction of acetoxy- and/or benzoyl-protected bis(saccharide)rubyrinconjugates such as 76a in Reaction Scheme M. Treatment of the acetoxyand/or benzoyl protected bis(saccharide)rubyrin 76a with KOH in methanolyields the corresponding deprotected bis(saccharide)rubyrin conjugate76b (Reaction Scheme M). ##STR4##

To prepare an ether linked bis(saccharide)rubyrin conjugate with theether linkages in the 4 and 17 positions on the rubyrin periphery, theappropriate acetoxy-protected diformyl bipyrrole can be prepared asshown in Reaction Scheme N. Reduction of the methyl esters of bipyrrole77 with borane-THF to the corresponding alcohols followed by protectionwith acetic anhydride provides the acetoxy-protected diol 79a. Standarddebenzylation, to afford diacid 79b, followed by Clezy formylation withTFA and triethylorthoformate yields the acetoxy-protected bipyrrole 80.Condensation of bipyrrole 80 with diacid 81, under rubyrin-formingconditions, provides the acetoxy-protected dihydroxy rubyrin 82a.Deprotection of the hydroxyl groups can be achieved by treatment withHCl in methanol to afford dihydroxyrubyrin 82b.

The dihydroxyrubyrin thus formed can be coupled with acetoxy- and/orbenzoyl-protected bromo-substituted saccharide units, such as structure75 (Reaction Scheme O), in methylene chloride with silver triflate andbarium carbonate to afford the acetoxy- and/or benzoyl-protectedbis(saccharide)rubyrin conjugate 84a, as shown in Reaction Scheme O.Treatment of the acetoxy and/or benzoyl protected bis(saccharide)rubyrin84a with KOH in methanol yields the corresponding deprotectedbis(saccharide)rubyrin conjugate 84b (Reaction Scheme O). ##STR5##

EXAMPLE X SYNTHESIS OF RUBYRIN ANALOGUES EMPLOYING HETEROATOMS

In addition to the above rather direct extensions, the skilled artisanwill appreciate that either bifuran and/or bithiophene subunits can beused in place of bipyrroles 11, 15, or 16 (FIG. 4). Using a subunitsubstitution, which will be known to those of skill in the art in lightof the present disclosure, will enable the preparation of heteroatomrubyrin-type compounds. These include, for example, those depicted bygeneralized structures 20-24 in FIG. 6, and structures 87, 92, 99 and104 of Reaction Schemes P through S, respectively.

In light of the present disclosure and the recent work in the sapphyrinarea, for example, Sessler et al, 1992⁷⁹, incorporated herein byreference, one of skill in the art would be able to prepare compounds ofthe general type shown in FIG. 6, and further exemplified in ReactionScheme P through Reaction Scheme S. In particular, it is important toappreciate that 5,5'-diformyl-2,2'-bifuran and5,5'-diformyl-2,2,-bithiophene are now readily available materials⁷⁹,80.Simple condensation of these materials, in a normal rubyrin-formingmanner, with a tetrapyrrolic fragment such as compound 14 of FIG. 4would be expected, therefore, to produce rubyrin analogues of generalclass 22 and 23, in which two of the normal six pyrrolic nitrogens arereplaced by sulfur or oxygen, respectively.

Reaction Schemes P through S represent examples of the synthesis ofrubyrin analogues employing heteroatoms. The starting materials for usein the syntheses represented in these reaction schemes, and hence theresultant products, may include various substituents, such as, forexample, H, alkyl, aryl, amino, hydroxyl, alkoxy, carboxy, carboxamide,ester, amide, sulfonato, hydroxy substituted alkyl, alkoxyl substitutedalkyl, carboxy substituted alkyl, amino substituted alkyl, sulfonatosubstituted alkyl, ester substituted alkyl, amide substituted alkyl,substituted aryl, substituted alkyl, substituted ester, substitutedether, or substituted amide groups.

A synthetic scheme depicting the synthesis of such a rubyrin in whichtwo of the normal six nitrogen heteroatoms are replaced by oxygen orsulfur is shown in Reaction Scheme P. The diacid tetrapyrrolic fragment85 can be condensed with the readily available diformyl bifuran ordiformyl bithiophene 86 under rubyrin-forming conditions, such as thosedescribed in Example I, to afford rubyrin analogue 87, where X can beeither O or S.

Rubyrin analogues in which two different pyrrolic nitrogens have beenreplaced by the heteroatoms oxygen or sulphur may also be prepared, forexample, as shown in Reaction Scheme Q. Condensation of bipyrrole 88with the acetoxy-activated furan or thiophene 89 to produce thetetracycle 90, followed by condensation with bipyrrole 91, under rubyrinforming conditions such as those described in Example I, produces the28π-electron rubyrin analogue 92, where X can be either oxygen orsulfur.

Reduction of bifuran or bithiophene fragments to the corresponding5,5'-bisacetoxymethyl derivatives will provide precursors that, inanalogy to the recent work in the heterosapphyrin area (Sessler et al,1992⁷⁹ incorporated herein by reference), will allow, followingcondensation with benzyl 3-ethyl-4-methyl-pyrrole-2-carboxylate andsubsequent hydrogenolysis (to remove the benzyl groups), the preparationof analogues of 14 in which the central tetraalkyl bipyrrole is replacedby either a bifuran or bithiophene moiety. Said analogues, followingcondensation with the appropriate diformyl-substituted bifuran orbithiophene, will allow, in turn, the preparation of rubyrin analoguesof generalized structure 20, 21, and 24, with the exact class ofcompound prepared depending on whether a bifuran (or bithiophene)containing tetracycle is condensed with a bifuran or bithiophenebicycle, as would be appreciated by one of skill in the art.

A synthetic scheme depicting the synthesis of a rubyrin analogue withfour of the nitrogen heteroatoms replaced by four oxygen or four sulfuratoms, or a combination of two oxygen and two sulphur atoms, is shown inReaction Scheme R. The readily available compound 93, where X can beeither O or S, can be reduced to the diol 94 by treatment with lithiumaluminum hydride and subsequently acetoxy-protected with aceticanhydride to provide compound 95. Acid catalyzed condensation of bifuranor bithiophene 95 with benzyl 3-ethyl-4-methyl-pyrrole-2-carboxylate inisopropanol provides the tetracycle 97a. Standard hydrogenolysis of 97awith H₂ and Pd/C affords diacid 97b, which can be condensed with thebicyclic fragment 98 (where Y=O or Y=S), under rubyrin formingconditions, to produce rubyrin analogue 99, where X and Y can separatelyand independently be oxygen or sulfur.

Rubyrin analogues in which all six of the nitrogen atoms have beenreplaced by oxygen or sulfur heteroatoms may also be synthesized, forexample, as shown in Reaction Scheme S. Condensation of the readilyavailable bifuran or bithiphene 100 with the readily availableacetoxy-activated furan or thiophene 101 affords the tetracycle 102.Condensing molecule 102 with a diformyl bifuran or diformyl bithiophene103, under rubyrin forming conditions such as those described in ExampleI, will give the 28π-electron macrocycle 104 where X, Y, and Z may beany combination of oxygen or sulfur, based on starting materials chosen.##STR6##

With such heteroatomic compounds, one may determine how changes in size,shape, and macrocycle denticity (the number of "coordinating" NH groups)affect monophosphate binding, and also define the basic structuralparameters associated with efficient through-membrane transport ofGMP-like species. Developing this theme will allow a determination ofhow these same changes in core structure are reflected in terms of anability (or lack thereof) to bind and transport other phosphorylatedspecies, including such important species as cyclic nucleotidemonophosphates (e.g. c-AMP), diphosphates (e.g. GDP), and triphosphates(e.g. ATP). Also, the extent to which these systems, and/or theirmonoprotonated derivatives, can act as receptors/carriers for varioushalides, including, of course, chloride anion may be investigated.

Furthermore, it is again contemplated that some or all of the β-pyrrolicpositions in these systems may be substituted with alkyl orfunctionalized alkyl substituents. Thus, a wide variety of structuresare conceived within the context of the basic synthetic methodologypresented in FIG. 4.

EXAMPLE XI RUBYRIN AND RUBYRIN ANALOGUES AS CHELATING RECEPTORS

To date, labile bis-zinc derivatives of 10a and 10b have been made.However, it is envisioned that the diprotonated rubyrin systems will actas effective receptors for a variety of anions, as is true for sapphyrinwhen protonated⁴,6, and possibly other cationic expandedporphyrins¹²⁻¹⁴. For instance, preliminary spectroscopic studies withsystem 10a indicate that diprotonated rubyrin can bind both fluoride andphosphate anions in a strong and non-labile manner.

Furthermore, the monoprotonated form of rubyrin 10a has been shown toact as an effective carrier for the through-membrane transport ofguanosine-5'-monophosphate in Pressman type model system. In light ofthese results, protonated rubyrins are proposed to be of use in avariety of molecular recognition applications, that are not within thepurview of normal tetrapyrrolic porphyrin chemistry.

Once a range of rubyrin analogues have been generated, for example, asdescribed herein in the foregoing detailed examples, the thermodynamicsand kinetics of anion binding under a range of conditions and with anarray of different anions may be determined. The structure and functionof the most promising rubyrin compounds may then be optimized such thatthey bind either phosphate-bearing nucleotides, or chloride ions, withhigh affinity and selectivity at neutral pH.

As a complement to structural studies, quantitative analyses may also beconducted. For example, the relevant pK_(a) ' values for variousrubyrins may be determined by employing the methods previously used todetermine the pK_(a) ' values for sapphyrin and anthraphyrin^(16h),6a.One should beware of possible "artifacts" arising from anion chelation,which can be avoided by using non-chelating buffers and solvents. Suchinitial studies should be followed by ones in which the various pK_(a) 'values are recorded in different solvents and in the presence ofdifferent anions such as hydrosulfate, bicarbonate, azide, cyanide,fluoride, bromide, and iodide, that are of biological relevance. Otherqualitative tests (e.g. UV/vis, FABMS) may also be used to reconfirmthat rubyrins do not form complexes under physiological conditions withNa⁺, K⁺, Ca²⁺, or other bio-cations. The reason for these latter studiesis that such cation complex formation could preclude efficient anionbinding and transport.

Once pK_(a) ' values are recorded, a second set of quantitative analysesmay be performed to determine the actual affinity constants for each andevery relevant receptor-to-anionic interaction. Thus, for instance,K_(s) for [H₆ Rub²⁺.GMP²⁻ ] formation in a variety of solvent systemsmay be measured in a similar manner to that for the hydrohalide salts ofsapphyrin and anthraphyrin^(16h). Standard methods as quantitativeUV/vis titrations and concentration dependent NMR chemical shiftanalyses may be used, along with more sophisticated techniques such asthose involving static and time-resolved fluorescence. The inventorshave determined that the latter methods offer considerable advantagesand are particularly useful for measuring high affinity constants (i.e.those in the K_(s) ≧10⁶ M⁻¹ range). These fluorescence-based methodsrequire highly colored materials with good singlet state emissioncharacteristics, but these criteria are clearly met by the rubyrins.

Quantitative kinetic studies may also be carried out and used todetermine whether the rate limiting step in GMP (or chloride) transportinvolves initial receptor-anion complex formation, through-membranecarrier-complex diffusion, product release, and/or rate of carrierback-diffusion. On- and off-rates for complex formation may be measured,for example, by dynamic NMR, UV/vis, or time-resolved fluorescence, in,e.g., simple water-saturated dichloromethane solutions. More preciseanalyses of receptor-mediated transport may also be made, again with amind to determining what are the dynamics of complexation anddecomplexation. The U-tube model system may be employed, and whenappropriate, more sophisticated membrane analogues such as mixedphosphatidylcholine-cholesterol liposomes can be used^(18a).

For the latter studies, it may prove most convenient to preparenucleotide or halide encapsulating liposomes and then determine thekinetics of anion extrusion as a function of carrier concentrationand/or external solution pH. Here again, either UV/vis or time-resolvedfluorescence analyses may be used. Here, it might prove necessary to adda specific fluorophore, such as 6-methoxy-N-(3-sulphopropyl)quinolinium(a halide selective reagent) to the outside phase so as to be able todetect small quantities of the anionic "escapees".

EXAMPLE XII RUBYRIN AND ANALOGUES AS CELLULAR ANION TRANSPORTERS

In preliminary transport screening studies, using a H₂ O--CH₂ Cl₂ --H₂ OPressman-type⁶,70,74 U-tube model system, rubyrin was found to becapable of effecting through-membrane transport of GMP and othernucleotides at near-neutral pH (i.e. in the pH 6.0 to 6.5 regime). Incontrast, sapphyrin was completely ineffective, and pentaphyrin mediatedonly very slow GMP transport, which was further, and unfortunately,subject to inhibition by chloride anions. Thus, it is clear that for anyconceivable phosphate chelation or recognition applications the rubyrinswill emerge as being vastly superior to either the pentaphrins orsapphyrins.

The effective transport by rubyrin, a larger, more basic system, mayderive from lower in-core NH⁺ -to-NH⁺ repulsions. In any event, theseproperties make rubyrin an ideal candidate for use in the delivery ofphosphorylated compounds such as antivirals. The ability of rubyrin toeffect nucleotide transport in a manner that is free of any chloride (orother halide) anion inhibition (even though this same material bindschloride anion quite effectively at very low pH and in the solid state)is particularly important.

Furthermore, the inventors determined that transport effected by thislatter rubyrin carrier could be made somewhat nucleobase selective byadding the appropriate complementary TIPS derivative to the organicmembrane phase. For example, in the presence of C-Tips(triisopropylsilyl-protected cytidine), the rate of rubyrin-mediated GMPtransport was observed to be substantially enhanced. Results from suchexploratory studies thus support not only the suggestion that it shouldbe possible to effect base-specific phosphate (or phosphonate) entitytransport under physiological conditions, but also the contention that,by synthetic "selection" or "fine tuning" it should be possible todesign rubyrin anion receptors that are selective for eitherphosphate-derived antivirals, or chloride anion, or both.

In further studies, those kinetic and thermodynamic factors thatmilitate both for and against rapid, selective transport ofphosphorylated nucleotides and nucleotide analogues can be preciselydetermined. Both the use of simple U-tube and more elaborate liposomaltest systems is contemplated.

EXAMPLE XIII USE OF RUBYRIN AND ANALOGUES AS THERAPEUTIC AGENTS

Rubyrin compounds of the present invention are contemplated to be of useas anion transporters in various embodiments relating to humantreatment. They are particularly contemplated for use as delivery agentsfor antiviral compounds and may thus be employed to combat a variety ofdiseases including AIDS, herpes, hepatitis and measles. Rubyrincompounds optimized for chloride transport are also contemplated for usein the treatment of cystic fibrosis.

In developing the rubyrin compounds of the present invention fortherapeutic use as anti-viral transporters, in vitro tests will first beconducted. These will follow protocols similar to those used earlier toscreen the photodynamic antiviral activity of sapphyrin and severalother expanded porphyrins^(18b). In brief, a monolayer of Vero cellswill be infected with HSV-1, coated with an overlay culture medium, andthen exposed to various relative and absolute concentrations of bothputative carrier and known active antiviral. Then, following incubationat 37° C., adjuvant efficacy will be determined by counting the numberof plaque forming units (PFU) obtained in the presence and absence of agiven carrier.

Following such in vitro tests, the activity of promising rubyrinreceptors will be followed-up, for example, in anti-HIV screens, andthen in in vivo animal studies. These studies will be conductedaccording to the standard practice for such animal trials, the executionof which will be known to those of skill in the art.

During the animal trial stage, the rubyrin compounds, whether used inantiviral delivery, or for chloride transport in cystic fibrosistreatment, may be modified further if required. They might, forinstance, be modified to overcome poor water solubility orsusceptibility to in vivo degradation. Alternatively, if such problemsoccur, the rubyrins could be enveloped within a bio-compatible liposome(made, e.g. from Cremophor)⁷³ and then administered intravenously. Suchan approach has previously resulted in good in vivo murineadenocarcinoma photodynamic tumor killing with a water-insolubletexaphyrin-type expanded porphyrin⁷⁴. The "Trojan Horse" method⁷⁵ couldalso be employed to deliver the rubyrin antiviral carrier in vivo to thedesired locus of biological activity. Here, the idea would be to usenon-infectious viral membrane material to produce liposomes and then usethese in turn as transport vehicles to get the putative carrier to thesite of cellular infectivity.

Naturally, toxicity studies will also be carried out at this stage. Themethods for determining both acute and chronic toxicity will be known tothose of skill in the art. Available evidence indicates that rubyrins,like sapphyrins, will be relatively nontoxic. Toxicity can beinvestigated in relation to solubility, net charge at physiologic pH,and changes in appended β-pyrrolic and/or meso substituents.

Furthermore, the phosphate-binding rubyrin compounds of the presentinvention may act as receptors and transporters for other biologicallyimportant molecules with negative charges, particularly, polynucleicacids such as DNA, RNA and oligonucleotides. Another dimension to theinvention, therefore, concerns the possibility of using rubyrins in thetransport of DNA molecules, such as antisense DNA constructs, into cellsfor use in so-called gene therapy programs.

Normal cellular uptake of negatively-charged DNA is known to be limited.Current in vitro methods rely on severe cellular modifications whichoften cause excessive cell damage⁷⁶, and as a result, are not viable invivo. The "coating" of nucleic acid phosphate groups with rubyrins, thusrendering them suitable for diffusional uptake in vivo, is thereforevery attractive and even has implications for chromosomal gene therapy.

While the compositions and methods of this invention have been describedin terms of preferred embodiments, it will be apparent to those of skillin the art that variations may be applied to the composition, methodsand in the steps or in the sequence of steps of the method describedherein without departing from the concept, spirit and scope of theinvention. More specifically, it will be apparent that certain agentswhich are both chemically and physiologically related may be substitutedfor the agents described herein while the same or similar results wouldbe achieved. All such similar substitutes and modifications apparent tothose skilled in the art are deemed to be within the spirit, scope andconcept of the invention as defined by the appended claims.

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What is claimed is:
 1. A macrocycle with one of the followingstructures: ##STR7## wherein: A₁ and A₂ are nitrogen, oxygen or sulphur;and the substituents R₁, R₂, R₃, R₄, R₅, and R₆ and X₁, X₂, X₃, and X₄are independently in each occurrence H, alkyl, aryl, amino, hydroxyl,alkoxy, carboxy, carboxamide, ester, amide, sulfonate, hydroxysubstituted alkyl, alkoxyl substituted alkyl, carboxy substituted alkyl,ester substituted alkyl, amide substituted alkyl, amino substitutedaryl, ester substituted aryl, ether substituted aryl, alkoxy substitutedaryl, carboxy substituted aryl, amide substituted aryl, thio substitutedester, phospho substituted ester, amino substituted ester, or are of theformula (CH₂)_(n) --A--(CH₂)_(m) --B, wherein A is CH₂, O, S, NH, orNR₇, wherein R₇ is any of the groups listed above, n and m are integers<10 or zero, and B includes a nucleobase or saccharide moiety.
 2. Themacrocycle of claim 1, wherein the macrocycle is either singly or doublyprotonated.
 3. The macrocycle of claim 2, wherein the macrocycle has astructure in accordance with structure I.
 4. The macrocycle of claim 2,wherein the macrocycle has a structure in accordance with structure II.5. The macrocycle of claim 2, wherein the macrocycle has a structure inaccordance with structure III.
 6. The macrocycle of claim 5, wherein themacrocycle is either singly, doubly, triply, or four-fold protonated. 7.The macrocycle of claim 1, wherein A₁ and A₂ are nitrogen.
 8. Themacrocycle of claim 1, wherein A₁ and A₂ are oxygen.
 9. The macrocycleof claim 1, wherein A₁ and A₂ are sulphur.
 10. The macrocycle of claim1, wherein either A₁ or A₂ is nitrogen and the other is oxygen.
 11. Themacrocycle of claim 1, wherein either A₁ or A₂ is nitrogen and the otheris sulphur.
 12. The macrocycle of claim 1, wherein either A₁ or A₂ isoxygen and the other is sulphur.
 13. The macrocycle of claim 1, whereinB is a nucleobase and the nucleobase is a purine or pyrimidine, or ananalog thereof.
 14. The macrocycle of claim 13, wherein the purine orpyrimidine or analog thereof comprises an antimetabolic, antienzymatic,antitumor, anticellular, antiproliferative or antiviral purine orpyrimidine analog selected from Table I.
 15. The macrocycle of claim 14,wherein the purine or pyrimidine analog comprises an antiviral,antimetabolic or antienzymatic compound.
 16. The macrocycle of claim 14,wherein the purine or pyrimidine analog comprises an antitumor compound.17. The macrocycle of claim 1, wherein B is a nucleobase and thenucleobase is cytosine, guanine, thymidine, adenine, uridine or inosine.18. The macrocycle of claim 1, wherein B is a nucleobase and thenucleobase is selected from Table I.
 19. The macrocycle of claim 1,wherein B is a nucleobase and the nucleobase includes a protecting groupon an amino group of the nucleobase, wherein the protecting group is9-fluorenylmethylcarbonyl, benzyloxycarbonyl,4-methoxyphenacyclocarbonyl, t-butyloxycarbonyl, 1-adamantyloxycarbonyl,benzoyl, N-triphenylmethyl, N-di-(4methoxyphenyl)phenylmethyl.
 20. Themacrocycle of claim 1, wherein B is a nucleobase and the nucleobase isan antimetabolite.
 21. The macrocycle of claim 20, wherein theantimetabolite is FU, AraC, AZT, ddI, xylo-GMP, Ara-AMP, PFA or LOMDP.22. The macrocycle of claim 1, further defined as having a structure ofFIG. 10 and wherein the macrocycle is mono-substituted with nucleobase.23. The macrocycle of claim 1, further defined as having a structure ofFIG. 10, wherein the macrocycle is disubstituted with nucleobase. 24.The macrocycle of claim 1, wherein B is a saccharide moiety.
 25. Themacrocycle of claim 24, wherein the saccharide moiety is a sugar, sugarderivative, or polysaccharide.
 26. The macrocycle of claim 25, whereinthe sugar or sugar derivative has a phosphate, methyl or amino group.27. The macrocycle of claim 24, wherein the saccharide moiety is ribose,arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose,idose, galactose, talose, ribulose, xylulose, psicose, fructose,sorbose, tagatose, fucose, methylglucoside, glucose-6-phosphate,N-acetylgalactosamine, N-acetylglucosamine, or sialic acid.
 28. Themacrocycle of claim 24, wherein the saccharide moiety is glucose,galactose, galactosamine, glucosamine or mannose.
 29. A macrocycle withone of the following structures: ##STR8## wherein: A₁ and A₂ arenitrogen, oxygen or sulphur; and the substituents R₁, R₂, R₃, R₄, R₅,and R₆ and X₁, X₂, X₃, and X₄ are independently in each occurrence H,alkyl, aryl, amino, hydroxyl, alkoxy, carboxy, carboxamide, ester,amide, sulfonate, hydroxy substituted alkyl, alkoxyl substituted alkyl,carboxy substituted alkyl, ester substituted alkyl, amide substitutedalkyl, amino substituted aryl, ester substituted aryl, ether substitutedaryl, alkoxy substituted aryl, carboxy substituted aryl, amidesubstituted aryl, thio substituted ester, phospho substituted ester,amino substituted ester, or are of the formula (CH₂)_(n) --A--(CH₂)_(m)--B, wherein A is CH₂, O, S, NH, or NR₇, wherein R₇ is any of the groupslisted above, n and m are integers <10 or zero, and B includes a metalchelating group, alkylating agent, steroid, steroid derivative, aminoacid, peptide, polypeptide, a rubyrin molecule, rubyrin derivatives, orpolymeric rubyrin, sapphyrin or texaphyrin or polymers or derivativesthereof, polymer matrix or solid support.
 30. The macrocycle of claim29, wherein B is a metal chelating agent.
 31. The macrocycle of claim29, wherein B is asteroid or steroid derivative.
 32. The macrocycle ofclaim 29, wherein B is an amino acid.
 33. The macrocycle of claim 29,wherein B is a peptide or polypeptide.
 34. The macrocycle of claim 29,wherein B is a rubyrin molecule, rubyrin derivatives or polymericrubyrin.
 35. The macrocycle of claim 29, wherein B is a sapphyrin ortexaphyrin or polymers or derivatives thereof.
 36. The macrocycle ofclaim 29, wherein B is a polymer matrix or solid support.